In this study, we used laser capture microdissection (LCM) and microarray hybridization technolog... more In this study, we used laser capture microdissection (LCM) and microarray hybridization technology to compare the gene expression profiles of mouse embryonic days 10 and 12 lenses (E10 and E12). Lens cells of C57/BL6 mouse embryos at E10 and E12 were harvested using the PixCell II LCM System. Total RNA was extracted, amplified, labeled, and hybridized to the 430 2.0 mouse chip (Affymetrix) according to the manufacturer's instructions. Data extracted from the images were analyzed using different software programs. Regulated expression of selected genes was confirmed by real-time PCR (RT-PCR). Analysis of the microarray data from E10 and E12 lenses identified 1,573 genes that showed a two fold or greater change in expression level. Among these 1,573 genes, 956 genes were downregulated and 617 were upregulated in E12 lenses. In addition to the upregulated expression of beta- and gamma-crystallin genes, genes that regulate the cell cycle showed significant changes of gene expression...
In conventional neurons, Ca2+ enters presynaptic terminals during an action potential and its inc... more In conventional neurons, Ca2+ enters presynaptic terminals during an action potential and its increased local concentration triggers transient exocytosis. In contrast, vertebrate photoreceptors are nonspiking neurons that maintain sustained depolarization and neurotransmitter release from ribbon synapses in darkness and produce light-dependent graded hyperpolarizing responses. Rods transmit single photon responses with high fidelity, whereas cones are less sensitive and exhibit faster response kinetics. These differences are likely due to variations in presynaptic Ca2+ dynamics. Metabolic coupling and cross-talk between mitochondria, endoplasmic reticulum (ER), plasma membrane Ca2+ ATPase (PMCA), and Na+-Ca2+ exchanger (NCX) coordinately control presynaptic ATP production and Ca2+ dynamics. The goal of our structural and functional studies was to determine the spatiotemporal regulation of ATP and Ca2+ dynamics in rod spherules and cone pedicles. Central retina tissue from C57BL/6 mi...
Purpose Postnatal lead exposure produces rod-selective and Bax-mediated apoptosis, decreased scot... more Purpose Postnatal lead exposure produces rod-selective and Bax-mediated apoptosis, decreased scotopic electroretinograms (ERGs), and scotopic and mesopic vision deficits in humans and/or experimental animals. Rod, but not cone, inner segment mitochondria were considered the primary site of action. However, photoreceptor synaptic mitochondria were not examined. Thus, our experiments investigated the structural and functional effects of environmentally relevant postnatal lead exposure on rod spherule and cone pedicle mitochondria and whether Bcl-xL overexpression provided neuroprotection. Methods C57BL/6N mice pups were exposed to lead only during lactation via dams drinking water containing lead acetate. The blood [Pb] at weaning was 20.6±4.7 µg/dl, which decreased to the control value by 2 months. To assess synaptic mitochondrial structural differences and vulnerability to lead exposure, wild-type and transgenic mice overexpressing Bcl-xL in photoreceptors were used. Electron micros...
PURPOSE Comparative studies of structure related to function offer a promising means of understan... more PURPOSE Comparative studies of structure related to function offer a promising means of understanding the significance of differences in cytoarchitecture. Mitochondrial crista structure is linked tightly to mitochondrial function. Non-foveal cone photoreceptors of primates contain considerably more inner segment mitochondria and have higher oxidative enzyme activity than do rods. In addition, it is suggested that light-adapted cones have a higher aerobic ATP demand than light-adapted rods. Therefore, we investigated the oxidative metabolism and three-dimensional membrane architecture of mouse rod and cone inner segment mitochondria. METHODS We determined the number, size, cytochrome c oxidase (CO) reactivity, and membrane architecture of rod and middle wavelength-sensitive (M) cone inner segment mitochondria from 21 day old light-adapted C57BL/6 mice using conventional electron microscopy and the three-dimensional approach of single- and double-tilt electron microscope tomography. F...
Studies of neuronal development in the retina often examine the stages of proliferation, differen... more Studies of neuronal development in the retina often examine the stages of proliferation, differentiation, and synaptic development, albeit independently. Our goal was to determine if a known neurotoxicant insult to a population of retinal progenitor cells (RPCs) would affect their eventual differentiation and synaptic development. To that end, we used our previously published human equivalent murine model of low-level gestational lead exposure (GLE). Children and animals with GLE exhibit increased scotopic electroretinogram a- and b-waves. Adult mice with GLE exhibit an increased number of late-born RPCs, a prolonged period of RPC proliferation, and an increased number of late-born rod photoreceptors and rod and cone bipolar cells (BCs), with no change in the number of late-born Müller glial cells or early-born neurons. The specific aims of this study were to determine whether increased and prolonged RPC proliferation alters the spatiotemporal differentiation and synaptic developmen...
Purpose: Comparative studies of structure related to function offer a promising means of understa... more Purpose: Comparative studies of structure related to function offer a promising means of understanding the significance of differences in cytoarchitecture. Mitochondrial crista structure is linked tightly to mitochondrial function. Non-foveal cone photoreceptors of primates contain considerably more inner segment mitochondria and have higher oxidative enzyme activity than do rods. In addition, it is suggested that light-adapted cones have a higher aerobic ATP demand than light-adapted rods. Therefore, we investigated the oxidative metabolism and three-dimensional membrane architecture of mouse rod and cone inner segment mitochondria. Methods: We determined the number, size, cytochrome c oxidase (CO) reactivity, and membrane architecture of rod and middle wavelength-sensitive (M) cone inner segment mitochondria from 21 day old light-adapted C57BL/6 mice using conventional electron microscopy and the three-dimensional approach of single- and double-tilt electron microscope tomography. Fourteen different measurements of mitochondrial substructures were analyzed. Photoreceptor oxygen consumption was determined in dark- and light-adapted retinas. Results: Rod and cone mitochondria displayed an orthodox conformation. Cone inner segments, compared to rods, contained 2-fold more mitochondria and were more CO reactive. Rod and cone mitochondria had similar outer-inner membrane width, contact site width, diameter and density, crista width, number of cristae/volume, number of cristae segments/volume, and fraction of cristae with multiple segments. In contrast, cone mitochondria had narrower crista junctions, greater cristae connectivity, and approximately 3-fold more cristae membrane surface area compared to rods. The increased cristae membrane surface area in cones was accomplished by connecting more cristae segments together, rather than by creating more cristae. Conclusions: These results demonstrate that middle wavelength (M) cones have a different bioenergetic signature than do rods and suggest that the aerobic ATP demand and production is greater in light-adapted cones than in light-adapted rods. Cones utilize two complimentary strategies to increase their aerobic ATP production: increase the number of mitochondria and increase the cristae surface membrane area. The greater ATP generation by cones may also provide increased protection against metabolic insults and apoptosis compared to rods.
Perovskite compounds, including lead-lanthanum-zirconium titanate (PLZT), have wide technological... more Perovskite compounds, including lead-lanthanum-zirconium titanate (PLZT), have wide technological application because of their unique physical properties. The use of PLZT in neuro-prosthetic systems, such as retinal implants, has been discussed in a number of publications. Since inorganic lead is a retinotoxic compound that produces retinal degeneration, the long-term stability of PLZT in aqueous biological solutions must be determined. We evaluated the stability and effects of prolonged immersion of a PLZT-coated crystal in a buffered balanced salt solution. Scanning Electron Microscopy and Electron Dispersive Spectroscopy (EDS) using a JEOL JSM 5410 microscope equipped with EDS were utilized to evaluate the samples before and after prolonged immersion. We found that lead and other constituents of PLZT leached into the surrounding aqueous medium. By comparing the unit cell of PLZT with that of CaTiO(3), which has been found to react with aqueous fluids, Lead is in the same site in PLZT as Ca is in CaTiO(3). It is thus reasonable that PLZT will react with aqueous solutions. The results suggest that PLZT must either be coated with a protective layer or is not appropriate for long-term in vivo or in vitro biological applications.
Test the hypothesis that the development of mammalian rod outer segments (ROS) varies with retina... more Test the hypothesis that the development of mammalian rod outer segments (ROS) varies with retinal eccentricity. During the period of photoreceptor cell development, ROS lengths, opsin mRNA and (rhod)opsin were measured in central and peripheral retina of cows and pigmented rats. Published ROS length and/or rhodopsin data from albino rats, cows and monkeys were re-analyzed. Logistic growth curves were fitted to the newly obtained and published data. Within a species, growth in central and peripheral regions was compared. The logistic growth curves fit all the data well and provide an excellent view of the developmental increases in ROS length, opsin mRNA and (rhod)opsin in each retinal region. Within a species, the growth curves for ROS length, opsin mRNA and (rhod)opsin concentration are superimposable. The age at which ROS length reaches 50% of its adult value is invariant with eccentricity. An exception to this pattern is the simian parafoveal ROS, which appears to have a delayed course of development. The hypothesis is disproved. Unlike rod photoreceptor cell genesis, ROS development is invariant with retinal eccentricity. Primate parafoveal ROS appear to have a different pattern of development.
Low-to-moderate level developmental and adult lead exposure produces retinal dysfunction and/or d... more Low-to-moderate level developmental and adult lead exposure produces retinal dysfunction and/or degeneration in humans and experimental animals. Although high level in vivo or in vitro lead disrupts blood-brain-barrier tight junctions and increases its permeability, the blood-retinal-barrier (BRB) has not been examined. There were four overall goals. First, generate environmentally relevant dose-response models of short-term lead exposure in adult rats. Second, assess retinal histology and functional integrity of the BRB. Third, investigate the transmembrane proteins occludin and claudin-5 as targets mediating the increased BRB permeability. Fourth, examine the contribution of the PI3K-Akt signaling pathway as a mechanism underlying increased BRB permeability. Young adult rats were given water, 0.01% or 0.02% lead drinking solutions for six weeks. In control, 0.01% and 0.02% groups the six week mean blood [Pb] were 1, 12.5 and 19μg/dl, respectively. We employed histology, stereology...
The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utiliza... more The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL...
A fundamental principle governing organ size and function is the fine balance between cell prolif... more A fundamental principle governing organ size and function is the fine balance between cell proliferation and cell differentiation. Here, we identify RONIN (THAP11) as a key transcriptional regulator of retinal progenitor cell (RPC) proliferation. RPC-specific loss of Ronin results in a phenotype strikingly similar to that resulting from the G1- to S-phase arrest and photoreceptor degeneration observed in the Cyclin D1 null mutants. However, we determined that, rather than regulating canonical cell-cycle genes, RONIN regulates a cohort of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Coincidentally, with premature cell-cycle exit, Ronin mutants exhibited deficient ETC activity, reduced ATP levels, and increased oxidative stress that we ascribe to specific loss of subunits within complexes I, III, and IV. These data implicate RONIN as a positive regulator of mitochondrial gene...
Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited,... more Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited, and disease-, injury- and chemical-induced retinal degeneration. Our aims were three-fold: 1) to determine the kinetics of rod apoptosis and Ca2+ overload in Pde6b9rd1) mice and developmentally lead-exposed rats, 2) to establish a pathophysiologically-relevant model of Ca2+ overload/rod-selective apoptosis in isolated rat retina and 3) to examine different mechanistic based neuroprotective strategies that would abrogate or mollify rod Ca2+ overload/apoptosis. Retinal morphometry and elemental calcium content ([Ca]) determined the kinetics of rod apoptosis and Ca2+ overload. A multiparametric analysis of apoptosis including rod [Ca], a live/dead assay, rod oxygen consumption, cytochrome c immunoblots and caspase assays was combined with pharmacological studies of an isolated rat retinal model of rod-selective Ca2+ overload/apoptosis. Ca2+ overload preceded rod apoptosis in mice and rats,...
To determine the dose-response relationship between blood lead concentration ([PbB]) and scotopic... more To determine the dose-response relationship between blood lead concentration ([PbB]) and scotopic ERG amplitude in 7- to 10-year-old children with lifetime lead exposure. Full-field flash scotopic ERGs were recorded over a 4-log-unit range in 45 dark-adapted children with normal visual acuity. [PbB] was measured throughout pregnancy and postnatal development, and the subjects' [PbB] levels were grouped at each age by tertiles. The median [PbB] during pregnancy was, from lowest to highest tertile, 2.5 to 5.0, 7.5 to 9.0, and 14.0 to 16.5 microg/dL, and after birth was 4.0 to 8.0, 6.0 to 14.5, and 7.5 to 21.0 microg/dL. Only maternal [PbB] at 12 weeks of pregnancy showed a significant dose-response relationship with the ERG measures, so that with increasing [PbB] there were significant increases in leading-edge a-wave amplitude, peak a-wave amplitude, and b-wave amplitude and sensitivity, with no changes in implicit times. Data analyses showed that children whose mothers had [PbB]...
All-trans retinoic acid (ATRA) or 9-cis retinoic acid (9CRA), added to dissociated developing neu... more All-trans retinoic acid (ATRA) or 9-cis retinoic acid (9CRA), added to dissociated developing neural retinal cells, induces progenitor cells to adopt the rod cell's fate. Retinoic acid (RA) also produces apoptotic cell death in developing tissues. The effects of retinoids on mouse retinal development were examined. Retinas were explanted on postnatal day (PN)1 and cultured with or without the retinal pigment epithelium (RPE) attached. Retinas were cultured for 3 weeks in the absence or presence of 100 or 500 nM ATRA or 9CRA. Morphologic development and apoptotic cell death were examined using cell-specific immunocytochemical markers, the TdT-dUTP terminal nick-end labeling (TUNEL) method, and a caspase assay. Retinal explants, with and without RPE, had similar age-dependent increases in opsin expression. In contrast, explants with RPE had less apoptosis during the first week than retinas without RPE. In explants with RPE, ATRA or 9CRA produced rod-selective apoptotic cell death ...
The promoter region of the rod-specific beta subunit of cGMP PDE (beta-PDE) and opsin genes conta... more The promoter region of the rod-specific beta subunit of cGMP PDE (beta-PDE) and opsin genes contains highly conserved cis-acting elements, which include an AP-1 and/or Nrl response element (NRE: An extended AP-1 like sequence). Transactivation of AP-1 or NRE appears necessary to drive expression of these rod-specific genes during adulthood, however, their role during development is relatively unknown. Therefore, we determined the spatial and temporal relationships between rod morphological and functional development, rod-specific gene expression, and expression of the bZIP transcription factors c-fos, junD and Nrl. Retinas from 0-45 day old (PN0-45) dark- and light-adapted Long-Evans rats were used. Morphological development was monitored by light and electron microscopy. Whole retinal trypsin-activated cGMP-PDE activity and rhodopsin content were measured biochemically. The expression of opsin, beta-PDE, c-fos, junD and Nrl mRNAs were determined by Northern blot analysis. The cellu...
METHODS. Retinal function was evaluated in dark-adapted mice by full-field electroretinograms (ER... more METHODS. Retinal function was evaluated in dark-adapted mice by full-field electroretinograms (ERGs) over more than 6 log units of intensity. Retinal morphology was studied by light- and electron microscopy. Arrestin and the heat shock protein 70 (Hsp70) were detected by Western blot analysis. The rhodopsin content and the kinetics of rhodopsin regeneration were determined in retinal extracts. RESULTS. Although the
In this study, we used laser capture microdissection (LCM) and microarray hybridization technolog... more In this study, we used laser capture microdissection (LCM) and microarray hybridization technology to compare the gene expression profiles of mouse embryonic days 10 and 12 lenses (E10 and E12). Lens cells of C57/BL6 mouse embryos at E10 and E12 were harvested using the PixCell II LCM System. Total RNA was extracted, amplified, labeled, and hybridized to the 430 2.0 mouse chip (Affymetrix) according to the manufacturer's instructions. Data extracted from the images were analyzed using different software programs. Regulated expression of selected genes was confirmed by real-time PCR (RT-PCR). Analysis of the microarray data from E10 and E12 lenses identified 1,573 genes that showed a two fold or greater change in expression level. Among these 1,573 genes, 956 genes were downregulated and 617 were upregulated in E12 lenses. In addition to the upregulated expression of beta- and gamma-crystallin genes, genes that regulate the cell cycle showed significant changes of gene expression...
In conventional neurons, Ca2+ enters presynaptic terminals during an action potential and its inc... more In conventional neurons, Ca2+ enters presynaptic terminals during an action potential and its increased local concentration triggers transient exocytosis. In contrast, vertebrate photoreceptors are nonspiking neurons that maintain sustained depolarization and neurotransmitter release from ribbon synapses in darkness and produce light-dependent graded hyperpolarizing responses. Rods transmit single photon responses with high fidelity, whereas cones are less sensitive and exhibit faster response kinetics. These differences are likely due to variations in presynaptic Ca2+ dynamics. Metabolic coupling and cross-talk between mitochondria, endoplasmic reticulum (ER), plasma membrane Ca2+ ATPase (PMCA), and Na+-Ca2+ exchanger (NCX) coordinately control presynaptic ATP production and Ca2+ dynamics. The goal of our structural and functional studies was to determine the spatiotemporal regulation of ATP and Ca2+ dynamics in rod spherules and cone pedicles. Central retina tissue from C57BL/6 mi...
Purpose Postnatal lead exposure produces rod-selective and Bax-mediated apoptosis, decreased scot... more Purpose Postnatal lead exposure produces rod-selective and Bax-mediated apoptosis, decreased scotopic electroretinograms (ERGs), and scotopic and mesopic vision deficits in humans and/or experimental animals. Rod, but not cone, inner segment mitochondria were considered the primary site of action. However, photoreceptor synaptic mitochondria were not examined. Thus, our experiments investigated the structural and functional effects of environmentally relevant postnatal lead exposure on rod spherule and cone pedicle mitochondria and whether Bcl-xL overexpression provided neuroprotection. Methods C57BL/6N mice pups were exposed to lead only during lactation via dams drinking water containing lead acetate. The blood [Pb] at weaning was 20.6±4.7 µg/dl, which decreased to the control value by 2 months. To assess synaptic mitochondrial structural differences and vulnerability to lead exposure, wild-type and transgenic mice overexpressing Bcl-xL in photoreceptors were used. Electron micros...
PURPOSE Comparative studies of structure related to function offer a promising means of understan... more PURPOSE Comparative studies of structure related to function offer a promising means of understanding the significance of differences in cytoarchitecture. Mitochondrial crista structure is linked tightly to mitochondrial function. Non-foveal cone photoreceptors of primates contain considerably more inner segment mitochondria and have higher oxidative enzyme activity than do rods. In addition, it is suggested that light-adapted cones have a higher aerobic ATP demand than light-adapted rods. Therefore, we investigated the oxidative metabolism and three-dimensional membrane architecture of mouse rod and cone inner segment mitochondria. METHODS We determined the number, size, cytochrome c oxidase (CO) reactivity, and membrane architecture of rod and middle wavelength-sensitive (M) cone inner segment mitochondria from 21 day old light-adapted C57BL/6 mice using conventional electron microscopy and the three-dimensional approach of single- and double-tilt electron microscope tomography. F...
Studies of neuronal development in the retina often examine the stages of proliferation, differen... more Studies of neuronal development in the retina often examine the stages of proliferation, differentiation, and synaptic development, albeit independently. Our goal was to determine if a known neurotoxicant insult to a population of retinal progenitor cells (RPCs) would affect their eventual differentiation and synaptic development. To that end, we used our previously published human equivalent murine model of low-level gestational lead exposure (GLE). Children and animals with GLE exhibit increased scotopic electroretinogram a- and b-waves. Adult mice with GLE exhibit an increased number of late-born RPCs, a prolonged period of RPC proliferation, and an increased number of late-born rod photoreceptors and rod and cone bipolar cells (BCs), with no change in the number of late-born Müller glial cells or early-born neurons. The specific aims of this study were to determine whether increased and prolonged RPC proliferation alters the spatiotemporal differentiation and synaptic developmen...
Purpose: Comparative studies of structure related to function offer a promising means of understa... more Purpose: Comparative studies of structure related to function offer a promising means of understanding the significance of differences in cytoarchitecture. Mitochondrial crista structure is linked tightly to mitochondrial function. Non-foveal cone photoreceptors of primates contain considerably more inner segment mitochondria and have higher oxidative enzyme activity than do rods. In addition, it is suggested that light-adapted cones have a higher aerobic ATP demand than light-adapted rods. Therefore, we investigated the oxidative metabolism and three-dimensional membrane architecture of mouse rod and cone inner segment mitochondria. Methods: We determined the number, size, cytochrome c oxidase (CO) reactivity, and membrane architecture of rod and middle wavelength-sensitive (M) cone inner segment mitochondria from 21 day old light-adapted C57BL/6 mice using conventional electron microscopy and the three-dimensional approach of single- and double-tilt electron microscope tomography. Fourteen different measurements of mitochondrial substructures were analyzed. Photoreceptor oxygen consumption was determined in dark- and light-adapted retinas. Results: Rod and cone mitochondria displayed an orthodox conformation. Cone inner segments, compared to rods, contained 2-fold more mitochondria and were more CO reactive. Rod and cone mitochondria had similar outer-inner membrane width, contact site width, diameter and density, crista width, number of cristae/volume, number of cristae segments/volume, and fraction of cristae with multiple segments. In contrast, cone mitochondria had narrower crista junctions, greater cristae connectivity, and approximately 3-fold more cristae membrane surface area compared to rods. The increased cristae membrane surface area in cones was accomplished by connecting more cristae segments together, rather than by creating more cristae. Conclusions: These results demonstrate that middle wavelength (M) cones have a different bioenergetic signature than do rods and suggest that the aerobic ATP demand and production is greater in light-adapted cones than in light-adapted rods. Cones utilize two complimentary strategies to increase their aerobic ATP production: increase the number of mitochondria and increase the cristae surface membrane area. The greater ATP generation by cones may also provide increased protection against metabolic insults and apoptosis compared to rods.
Perovskite compounds, including lead-lanthanum-zirconium titanate (PLZT), have wide technological... more Perovskite compounds, including lead-lanthanum-zirconium titanate (PLZT), have wide technological application because of their unique physical properties. The use of PLZT in neuro-prosthetic systems, such as retinal implants, has been discussed in a number of publications. Since inorganic lead is a retinotoxic compound that produces retinal degeneration, the long-term stability of PLZT in aqueous biological solutions must be determined. We evaluated the stability and effects of prolonged immersion of a PLZT-coated crystal in a buffered balanced salt solution. Scanning Electron Microscopy and Electron Dispersive Spectroscopy (EDS) using a JEOL JSM 5410 microscope equipped with EDS were utilized to evaluate the samples before and after prolonged immersion. We found that lead and other constituents of PLZT leached into the surrounding aqueous medium. By comparing the unit cell of PLZT with that of CaTiO(3), which has been found to react with aqueous fluids, Lead is in the same site in PLZT as Ca is in CaTiO(3). It is thus reasonable that PLZT will react with aqueous solutions. The results suggest that PLZT must either be coated with a protective layer or is not appropriate for long-term in vivo or in vitro biological applications.
Test the hypothesis that the development of mammalian rod outer segments (ROS) varies with retina... more Test the hypothesis that the development of mammalian rod outer segments (ROS) varies with retinal eccentricity. During the period of photoreceptor cell development, ROS lengths, opsin mRNA and (rhod)opsin were measured in central and peripheral retina of cows and pigmented rats. Published ROS length and/or rhodopsin data from albino rats, cows and monkeys were re-analyzed. Logistic growth curves were fitted to the newly obtained and published data. Within a species, growth in central and peripheral regions was compared. The logistic growth curves fit all the data well and provide an excellent view of the developmental increases in ROS length, opsin mRNA and (rhod)opsin in each retinal region. Within a species, the growth curves for ROS length, opsin mRNA and (rhod)opsin concentration are superimposable. The age at which ROS length reaches 50% of its adult value is invariant with eccentricity. An exception to this pattern is the simian parafoveal ROS, which appears to have a delayed course of development. The hypothesis is disproved. Unlike rod photoreceptor cell genesis, ROS development is invariant with retinal eccentricity. Primate parafoveal ROS appear to have a different pattern of development.
Low-to-moderate level developmental and adult lead exposure produces retinal dysfunction and/or d... more Low-to-moderate level developmental and adult lead exposure produces retinal dysfunction and/or degeneration in humans and experimental animals. Although high level in vivo or in vitro lead disrupts blood-brain-barrier tight junctions and increases its permeability, the blood-retinal-barrier (BRB) has not been examined. There were four overall goals. First, generate environmentally relevant dose-response models of short-term lead exposure in adult rats. Second, assess retinal histology and functional integrity of the BRB. Third, investigate the transmembrane proteins occludin and claudin-5 as targets mediating the increased BRB permeability. Fourth, examine the contribution of the PI3K-Akt signaling pathway as a mechanism underlying increased BRB permeability. Young adult rats were given water, 0.01% or 0.02% lead drinking solutions for six weeks. In control, 0.01% and 0.02% groups the six week mean blood [Pb] were 1, 12.5 and 19μg/dl, respectively. We employed histology, stereology...
The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utiliza... more The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL...
A fundamental principle governing organ size and function is the fine balance between cell prolif... more A fundamental principle governing organ size and function is the fine balance between cell proliferation and cell differentiation. Here, we identify RONIN (THAP11) as a key transcriptional regulator of retinal progenitor cell (RPC) proliferation. RPC-specific loss of Ronin results in a phenotype strikingly similar to that resulting from the G1- to S-phase arrest and photoreceptor degeneration observed in the Cyclin D1 null mutants. However, we determined that, rather than regulating canonical cell-cycle genes, RONIN regulates a cohort of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Coincidentally, with premature cell-cycle exit, Ronin mutants exhibited deficient ETC activity, reduced ATP levels, and increased oxidative stress that we ascribe to specific loss of subunits within complexes I, III, and IV. These data implicate RONIN as a positive regulator of mitochondrial gene...
Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited,... more Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited, and disease-, injury- and chemical-induced retinal degeneration. Our aims were three-fold: 1) to determine the kinetics of rod apoptosis and Ca2+ overload in Pde6b9rd1) mice and developmentally lead-exposed rats, 2) to establish a pathophysiologically-relevant model of Ca2+ overload/rod-selective apoptosis in isolated rat retina and 3) to examine different mechanistic based neuroprotective strategies that would abrogate or mollify rod Ca2+ overload/apoptosis. Retinal morphometry and elemental calcium content ([Ca]) determined the kinetics of rod apoptosis and Ca2+ overload. A multiparametric analysis of apoptosis including rod [Ca], a live/dead assay, rod oxygen consumption, cytochrome c immunoblots and caspase assays was combined with pharmacological studies of an isolated rat retinal model of rod-selective Ca2+ overload/apoptosis. Ca2+ overload preceded rod apoptosis in mice and rats,...
To determine the dose-response relationship between blood lead concentration ([PbB]) and scotopic... more To determine the dose-response relationship between blood lead concentration ([PbB]) and scotopic ERG amplitude in 7- to 10-year-old children with lifetime lead exposure. Full-field flash scotopic ERGs were recorded over a 4-log-unit range in 45 dark-adapted children with normal visual acuity. [PbB] was measured throughout pregnancy and postnatal development, and the subjects' [PbB] levels were grouped at each age by tertiles. The median [PbB] during pregnancy was, from lowest to highest tertile, 2.5 to 5.0, 7.5 to 9.0, and 14.0 to 16.5 microg/dL, and after birth was 4.0 to 8.0, 6.0 to 14.5, and 7.5 to 21.0 microg/dL. Only maternal [PbB] at 12 weeks of pregnancy showed a significant dose-response relationship with the ERG measures, so that with increasing [PbB] there were significant increases in leading-edge a-wave amplitude, peak a-wave amplitude, and b-wave amplitude and sensitivity, with no changes in implicit times. Data analyses showed that children whose mothers had [PbB]...
All-trans retinoic acid (ATRA) or 9-cis retinoic acid (9CRA), added to dissociated developing neu... more All-trans retinoic acid (ATRA) or 9-cis retinoic acid (9CRA), added to dissociated developing neural retinal cells, induces progenitor cells to adopt the rod cell's fate. Retinoic acid (RA) also produces apoptotic cell death in developing tissues. The effects of retinoids on mouse retinal development were examined. Retinas were explanted on postnatal day (PN)1 and cultured with or without the retinal pigment epithelium (RPE) attached. Retinas were cultured for 3 weeks in the absence or presence of 100 or 500 nM ATRA or 9CRA. Morphologic development and apoptotic cell death were examined using cell-specific immunocytochemical markers, the TdT-dUTP terminal nick-end labeling (TUNEL) method, and a caspase assay. Retinal explants, with and without RPE, had similar age-dependent increases in opsin expression. In contrast, explants with RPE had less apoptosis during the first week than retinas without RPE. In explants with RPE, ATRA or 9CRA produced rod-selective apoptotic cell death ...
The promoter region of the rod-specific beta subunit of cGMP PDE (beta-PDE) and opsin genes conta... more The promoter region of the rod-specific beta subunit of cGMP PDE (beta-PDE) and opsin genes contains highly conserved cis-acting elements, which include an AP-1 and/or Nrl response element (NRE: An extended AP-1 like sequence). Transactivation of AP-1 or NRE appears necessary to drive expression of these rod-specific genes during adulthood, however, their role during development is relatively unknown. Therefore, we determined the spatial and temporal relationships between rod morphological and functional development, rod-specific gene expression, and expression of the bZIP transcription factors c-fos, junD and Nrl. Retinas from 0-45 day old (PN0-45) dark- and light-adapted Long-Evans rats were used. Morphological development was monitored by light and electron microscopy. Whole retinal trypsin-activated cGMP-PDE activity and rhodopsin content were measured biochemically. The expression of opsin, beta-PDE, c-fos, junD and Nrl mRNAs were determined by Northern blot analysis. The cellu...
METHODS. Retinal function was evaluated in dark-adapted mice by full-field electroretinograms (ER... more METHODS. Retinal function was evaluated in dark-adapted mice by full-field electroretinograms (ERGs) over more than 6 log units of intensity. Retinal morphology was studied by light- and electron microscopy. Arrestin and the heat shock protein 70 (Hsp70) were detected by Western blot analysis. The rhodopsin content and the kinetics of rhodopsin regeneration were determined in retinal extracts. RESULTS. Although the
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Papers by Donald Fox