Browsing by Author "Johnson, Jerry E., Jr."
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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 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.