Browsing by Author "Fox, Donald A."
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Item Compartmental and Cellular Energy Metabolism in Adult and Developing Mouse Retina(2016-05) Rueda, Elda Maria; Fox, Donald A.; Frishman, Laura J.; Johnson, Jerry E., Jr.; Patel, Nimesh B.; Poché, Ross A.Purpose: There is a strong and direct relationship between bioenergetic metabolism and neuronal function during normal and pathophysiological conditions. Understanding these relationships is critical during development since different retinal regions undergo temporally-regulated cell proliferation, cell differentiation, synaptogenesis and synaptic refinement. The synthesis and maintenance of ATP and GTP (high energy phosphates: ~P) occurs via the regulation and integration of glycolysis/aerobic glycolysis (e.g., hexokinase, pyruvate kinase and lactate dehydrogenase: LDH), the TCA cycle (e.g., alpha ketoglutarate/oxoglutarate dehydrogenase, succinate thiokinase, succinate dehydrogenase, glutamate dehydrogenase), OXPHOS (e.g., COX4), and ~P transferring kinases (e.g., nucleoside diphosphate kinases, adenylate kinases and creatine kinases). Limited information is available about cell- and compartmental specific bioenergetics in developing and adult retina: especially between outer retina (photoreceptors: PRs) and inner retina. The overarching hypothesis is that the regulation of ~P synthesis and maintenance is age-, cell- and compartment-dependent. The goals of these biochemically-based studies were to characterize gene expression, protein expression, and enzymatic activity of key synthesizing and regulating enzymes of energy metabolism in adult and developing mouse retina. Methods: Embryonic, developing and adult C57BL/6N mice were used (3-7 retinas from 4-7 different mice from different litters per age). Homogenized whole retinas were used for Affymetrix microarray analysis and Western blots quantified by densitometry. Fixed-frozen retinas were used for semi-quantitative immunohistochemistry/laser scanning confocal microscopy, and LDH and COX activity studies: quantified by densitometry. Four non-adjacent central retina sections were used for each age and antibody or dye). ANOVAs and Student’s T-test were used for data analysis. Results: Adult retinas showed highly compartmentalized and graded expression and/or activity levels of isozymes of glycolysis/aerobic glycolysis, the TCA cycle, OXPHOS, and ~P transferring kinases. The outer, compared to inner, retina exhibited markedly higher aerobic glycolytic (LDH) and OXPHOS (COX) activity, and protein expression of HK, PK and COX4-1. PR inner segments and synaptic terminals, where 75% of mitochondria reside, had the highest LDH and COX activity and protein expression. The inner retina did not exhibit aerobic glycolytic capacity. Expression of TCA cycle anaplerosis/cataplerosis proteins was high in inner segments and moderate in the inner retina including Müller glial cells, indicating these compartments were supported by non-glucose derivatives. The ~P transferring kinases were differentially distributed among all compartments of retina. In developing retina, an age-dependent gene and expression and enzymatic activity of the aforementioned isoenzymes was observed. Retinal progenitor cells and the outer retina maintained high levels of LDH activity, whereas differentiating neurons increased their COX activity: especially in inner segments, synapses and the ganglion cell layer. Glycolysis and OXPHOS increased steadily with differentiation, whereas aerobic glycolysis and the TCA cycle was moderate and unchanged during retinal development. Conclusions: This study revealed that adult and developing retina express a number of genes related to ATP synthesizing and regulating enzymes found in muscle, liver, brain and/or highly proliferative cells (i.e. stem and cancer cells) that play significant roles in the cellular proliferation, cellular differentiation, cell development and cell-specific functional activity. The developing retina undergoes a metabolic reprogramming of aerobic glycolysis (LDH) and OXPHOS that results in the adult phenotype. The peak of these temporal changes correspond to the time of functional synaptic connection between PRs, bipolar cells and ganglion cells, and of eye opening. We propose that distinctive and characteristic metabolic changes occur when retinal progenitor cells exit the cell cycle and begin differentiation, and that an early increase in OXPHOS capacity is essential for neurons to acquire their identity and functionality. Together, these basic science results provide a platform for developing therapeutic strategies for neuroprotection, replacement and repair of retinal deficits/degeneration in a cell-specific manner: the goal of the NEI Audacious Goals Initiative.Item Electroretinographic studies in an animal model of fetal alcohol syndrome(1986) Katz, Laurence M.; Fox, Donald A.; Smith, Earl L., III; Levi, Dennis; Crawford, Jack M.It has been established beyond reasonable doubt that prenatal ethanol exposure is teratogenic to the developing fetus. Infants born to alcoholic women are frequently diagnosed as having the Fetal Alcohol Syndrome (FAS), which is primarily characterized by reduced birth and brain weight, a significant degree of mental retardation, and specific craniofacial dysmorphogenesis. The craniofacial anomalies include ocular manifestations such as microphthalmia, strabismus, epiphakia and amblyopia. These adverse effects of prenatal ethanol exposure on the anatomical development of the eye are well documented, however, its effect on the physiological function of the retina is unknown. The purpose of the present study was to identify and characterize functional retinal alterations resulting from ethanol exposure during the prenatal developmental period. Specifically, the objectives were to develop an animal (Rat) model of FAS, and to measure, using the electroretinogram (ERG), amplitude intensity functions (V-log I), temporal response functions, and adaptation functions under varying background levels of illumination. Single flash ERGs were measured under dark-adapted (DA) and light- adapted (LA) conditions, in the female offspring of Long-Evans hooded rats. One group was exposed to ethanol (ETOH 1) only during pregnancy, while a second group was exposed over an additional 10 day postnatal period (ETOH 2). Compared to controls the treatment offspring had reduced birth and organ weights, altered organ/body weight ratios and microphthalmia- all of these effects are indicators of FAS. During development these offspring continued to exhibit retarded growth. Under DA conditions, rod ERGs showed (i) a 1-log-unit increase in the absolute threshold for the b-wave in ETOH 1 and ETOH 2, (ii) an exposure-related decrease in the maximum amplitude of the a- and b-wave and (iii) an increase in the a-wave and b-wave latency only in ETOH 2. Under LA conditions, decreases in the b-wave V-Iog I function occurred all at background intensities, resulting in a non-uniform response compression. Increment-threshold functions were decreased at the dimmest background intensity using a low amplitude criterion, while using a higher criterion revealed an exposure-response decrease in sensitivity at the three lowest background intensities used. In addition, the prenatal ethanol exposure caused an altered sensitivity of DA following a bleach and depressed amplitude-recovery functions of the b-wave. However, no effect on the critical flicker frequency was observed. It is concluded that in utero ethanol exposure produced alterations in retinal adaptive mechanisms, "gain" mechanisms and temporal response properties. Ethanol exposure continued during the early postnatal period resulted in more profound changes in these measures. Possible biochemical mechanisms include, but are not limited to, reduced rhodopsin concentrations secondary to systemic vitamin A and zinc deficiency and changes in the extracellular potassium concentration due to retinal membrane alterations.Item Low-level human equivalent gestational lead exposure produces sex-specific motor and coordination abnormalities and late-onset obesity in year-old mice(Environmental Health Perspectives, 2008-03) Leasure, J. Leigh; Giddabasappa, Anand; Chaney, Shawnta Y.; Johnson, Jerry E., Jr.; Pothakos, Konstantinos; Lau, Yuen-Sum; Fox, Donald A.Background. Low-level developmental lead exposure is linked to cognitive and neurological disorders in children. However, the long-term effects of gestational lead exposure (GLE) have received little attention. Objectives. Our goals were to establish a murine model of human equivalent GLE and to determine dose–response effects on body weight, motor functions, and dopamine neurochemistry in year-old offspring. Methods. We exposed female C57BL/6 mice to water containing 0, 27 (low), 55 (moderate), or 109 ppm (high) of lead from 2 weeks prior to mating, throughout gestation, and until postnatal day 10 (PN10). Maternal and litter measures, blood lead concentrations ([BPb]), and body weights were obtained throughout the experiment. Locomotor behavior in the absence and presence of amphetamine, running wheel activity, rotarod test, and dopamine utilization were examined in year-old mice. Results. Peak [BPb] were < 1, ≤ 10, 24–27, and 33–42 μg/dL in control, low-, moderate- and high-dose GLE groups at PN0–10, respectively. Year-old male but not female GLE mice exhibited late-onset obesity. Similarly, we observed male-specific decreased spontaneous motor activity, increased amphetamine-induced motor activity, and decreased rotarod performance in year-old GLE mice. Levels of dopamine and its major metabolite were altered in year-old male mice, although only forebrain utilization increased. GLE-induced alterations were consistently larger in low-dose GLE mice. Conclusions. Our novel results show that GLE produced permanent male-specific deficits. The nonmonotonic dose-dependent responses showed that low-level GLE produced the most adverse effects. These data reinforce the idea that lifetime measures of dose–response toxicant exposure should be a component of the neurotoxic risk assessment process.Item Retinal development and age related degeneration following gestational lead exposure(2013-08) Chaney, Shawnta Y.; Fox, Donald A.; Eichberg, Joseph; Johnson, Jerry E., Jr.; Gunaratne, Preethi H.PURPOSE: Gestational lead exposure (GLE) increased and prolonged retinal progenitor cell proliferation in mice, resulting in a dose-dependent increase in two late-born retinal neurons: rod photoreceptors and bipolar cells. The present goals were to examine: 1) the spatiotemporal differentiation and functional development of these two cell types, 2) the profile of glutamatergic responses in developing retina prior to canonical synaptic function, and 3) the structural integrity of aging retinas after GLE. METHODS: RT-qPCR, immunohistochemistry, confocal microscopy, agmatine probe labeling, and pharmacological assays. RESULTS: GLE decreased the relative expression of rod specific genes at PN2; delayed the differentiation and functional development of rod photoreceptors and bipolar cells by 2-3 days; increased and prolonged the glutamatergic response of post-mitotic rod and bipolar precursors in the ventricular zone of developing retina; and increased and accelerated the age-related degeneration of rod photoreceptors and bipolar cells. CONCLUSIONS: Gestational exposure to environmental toxicants such as lead can produce differential age-dependent effects on the developing and aging retina. Furthermore, the lifespan effect of increased proliferation can result in degeneration later in life.Item Towards Mammalian Retinal Regeneration: Cell Signaling in Re-Differentiating Mouse Müller Glia and Transcriptional Regulation of the Axonal Guidance Receptor EPHA5(2016-05) Beach, Krista Marie; Otteson, Deborah C.; Frishman, Laura J.; Fox, Donald A.; Wang, StevenDamage to the neural retina or optic nerve results in irreversible blindness because the mammalian retina cannot regenerate. For stem cell therapies to regenerate a functional retina, multiple challenges must be overcome. These include identification of stem cell sources, targeted differentiation into particular retinal neuronal cell types, delivery to the damaged area, survival and integration into the remaining retinal tissue, and establishment of appropriate connections between cells. Müller glia have been proposed as potential retinal stem cells, but attempts to re-differentiate Müller glia into retinal neurons have shown poor yield and incomplete differentiation. In cultured retinal progenitor cells, differential activation of MAPK versus STAT3 cell signaling pathways by the cytokine ciliary neurotrophic factor (CNTF) can promote neuronal versus glial differentiation, respectively. Specific Aim 1 measured MAPK and STAT3 signaling and neuronal versus glial gene expression in a mouse Müller cell line during re-differentiation and in response to treatment with CNTF. At different stages of in vitro re-differentiation, ImM10 Müller glia activated neurogenic MAPK and did not change gliogenic STAT3 signaling pathways. Exogenous CNTF did not alter MAPK or STAT3 signaling or the final expression levels of neuronal and glial genes. Overall, ImM10 cells do not respond to CNTF as previously reported for primary Müller glia, and their neurogenic potential is not altered by CNTF. Another challenge facing regenerative therapies will be re-establishment of appropriate connections between the retina and the visual areas of the brain. The axonal guidance receptors EphA5 and EphA6 are expressed in gradients across the retina, and changes in their expression alter the location of retinal ganglion cell (RGC) synaptic termination zones. Therefore, new RGCs will need to express EphA5 and EphA6 at levels appropriate for their retinal location. Specific Aim 2 addressed the regulatory hierarchy controlling EphA5 and EphA6 expression in the mouse retina. POU4F2 is a RGC-specific transcription factor that when deleted results in aberrant RGC projections, suggesting potential defects in expression of axonal guidance genes. KLF16 is a recently-identified regulator of EphA5 that is expressed after POU4F2 in newly-differentiated RGCs, but KLF16’s regulation and spatial expression patterns in the retina have not been characterized. In wild-type mice, KLF16 expression was equal in nasal retina comparted to temporal retina. In Pou4f2 knockout mice, expression of EphA5, EphA6, and Klf16 was unchanged compared to wild-type mice. Unexpectedly, there was increased expression of a commonly used reference gene, β-2 microglobulin (B2m), which has been implicated in retinogeniculate synaptic remodeling. Thus POU4F2 may repress B2m expression, but does not regulate EphA5, EphA6, or Klf16 expression. In conclusion, in vitro differentiation of ImM10 Müller glia results in conflicting activation of MAPK and STAT3 signaling that may, in part, explain the limited neurogenic potential of this cell line. This emphasizes the necessity of finding alternative stem cell sources for regenerative therapies. In the Pou4f2-/- mouse, axonal misrouting is not a result of dysregulation of EphA5, EphA6, or Klf16, but may involve dysregulation of B2m. Further studies are needed to identify the transcriptional regulators of axonal guidance genes that could be exploited to promote axon outgrowth, guidance, and connectivity of stem cell-generated RGCs for development of regenerative therapies.Item TOWARDS RETINA REGENERATION: THE EPIGENETIC BASIS OF NEURONAL AND AXON GUIDANCE GENE REGULATION(2012-05) Petkova, Tihomira D. 1974-; Otteson, Deborah C.; McDermott, Alison M.; Wang, Steven; Gunaratne, Preethi H.; Fox, Donald A.Degenerative disorders, such as glaucoma are among the leading causes of irreversible blindness worldwide. With these disorders affecting a larger fraction of the population yearly, the need for regenerative retina and optic nerve therapy is self-evident. Increasing evidence suggests that Müller glia are potential stem cells in the adult mammalian retina but have a limited potential to differentiate into retinal ganglion cell (RGC). Understanding the mechanisms regulating expression of RGC specific and axon-guidance genes during development and in retinal stem cells is key for successful optic nerve regeneration. I proposed that dedifferentation of Müller glia to RGCs is restricted by silencing of RGC specific and axon guidance genes by chromatin remodeling mechanisms such as DNA methylation. The DNA methylation pattern of RGC determining gene Atoh7 and axon guidance genes EphA5 and EphB1 in Müller glia derived spheres were investigated prior to and following demethylation. Bisulfite sequencing (BS) showed that in ImM10 spheres, the promoters of these genes exhibited a high frequency of methylation and quantitative RT-PCR showed that the genes are not transcribed. Demethylation with 5-azadeoxycytidine (AzadC) resulted in a significant decrease of methylation at the gene promoters and expression of their mRNA. Priming ImM10 derived spheres in the presence of EGF and differentiating the cells in the presence of BDNF resulted in increased expression of pluripotent, RGC developmental and retinal pigment epithelium specific genes. These gene expression changes were associated with cell morphology changes consistent with that of cells of epithelial identity. Furthermore, I showed that DNA methylation is required for this BDNF driven morphological change. Additional evidence for the role of DNA methylation in the regulation of the retinal temporal-nasal gradient of EphA5 was found in BS analysis of the P0 mouse retina. In the nasal retina a modest, but statistically significant increase in methylation was correlated with lower levels of receptor mRNA expression compared to the temporal retina. The inverse relationship between EphA5 promoter methylation and mRNA expression is consistent with a role for DNA methylation in modulating the spatial patterns of EphA5 gene expression in the retina and in silencing EphA5 expression in ImM10 cells. In addition to regulation by DNA methylation, the EphA5 proximal promoter contains four predicted TCF/LEF binding sites, suggesting a potential role for WNT signaling in the transcriptional regulation of EphA5. In luciferase assays, activation of the canonical WNT signaling pathway increased the activity of mouse EphA5 promoter constructs in HEK293. WNT signaling activation increased expression of the endogenous EphA5 gene in retinal progenitor cells in vitro but failed to upregulate expression of the gene in retinal explants ex vivo, possibly due to the lack of one or several WNT signaling components in the P7 retina. Taken together these data provide the first evidence for a direct role of DNA methylation in the regulation of RGC specific genes. The combinated effects of BDNF treatment and DNA demethylation are consistent with epigenetic limitations on the proliferative and neurogenic potential of Müller glia in vitro. Reversing epigenetic silencing of neuronal genes combined with growth factor treatments to enhance neuronal survival offer a novel strategy for increasing neurogenesis from Müller-derived stem cells.