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Colin J Barnstable

Publications and source records attributed to Colin J Barnstable.

At least 19 recordsLinked to original sources
PubMed2006

A biphasic pattern of gene expression during mouse retina development.

BACKGROUND: Between embryonic day 12 and postnatal day 21, six major neuronal and one glia cell type are generated from multipotential progenitors in a characteristic sequence during mouse retina development. We investigated expression patterns of retina transcripts during the major embryonic and postnatal developmental stages to provide a systematic view of normal mouse retina development, RESULTS: A tissue-specific cDNA microarray was generated using a set of sequence non-redundant EST clones collected from mouse retina. Eleven stages of mouse retina, from embryonic day 12.5 (El2.5) to postnatal day 21 (PN21), were collected for RNA isolation. Non-amplified RNAs were labeled for microarray experiments and three sets of data were analyzed for significance, hierarchical relationships, and functional clustering. Six individual gene expression clusters were identified based on expression patterns of transcripts through retina development. Two developmental phases were clearly divided with postnatal day 5 (PN5) as a separate cluster. Among 4,180 transcripts that changed significantly during development, approximately 2/3 of the genes were expressed at high levels up until PN5 and then declined whereas the other 1/3 of the genes increased expression from PN5 and remained at the higher levels until at least PN21. Less than 1% of the genes observed showed a peak of expression between the two phases. Among the later increased population, only about 40% genes are correlated with rod photoreceptors, indicating that multiple cell types contributed to gene expression in this phase. Within the same functional classes, however, different gene populations were expressed in distinct developmental phases. A correlation coefficient analysis of gene expression during retina development between previous SAGE studies and this study was also carried out. CONCLUSION: This study provides a complementary genome-wide view of common gene dynamics and a broad molecular classification of mouse retina development. Different genes in the same functional clusters are expressed in the different developmental stages, suggesting that cells might change gene expression profiles from differentiation to maturation stages. We propose that large-scale changes in gene regulation during development are necessary for the final maturation and function of the retina.

Animals↗
PubMed2006

Molecular phylogeny of the antiangiogenic and neurotrophic serpin, pigment epithelium derived factor in vertebrates.

BACKGROUND: Pigment epithelium derived factor (PEDF), a member of the serpin family, regulates cell proliferation, promotes survival of neurons, and blocks growth of new blood vessels in mammals. Defining the molecular phylogeny of PEDF by bioinformatic analysis is one approach to understanding the link between its gene structure and its function in these biological processes. RESULTS: From a comprehensive search of available DNA databases we identified a single PEDF gene in all vertebrate species examined. These included four mammalian and six non-mammalian vertebrate species in which PEDF had not previously been described. A five gene cluster around PEDF was found in an approximate 100 kb region in mammals, birds, and amphibians. In ray-finned fish these genes are scattered over three chromosomes although only one PEDF gene was consistently found. The PEDF gene is absent in invertebrates including Drosophila melanogaster (D. melanogaster), Caenorhabditis elegans (C. elegans), and sea squirt (C. intestinalis). The PEDF gene is transcribed in all vertebrate phyla, suggesting it is biologically active throughout vertebrate evolution. The multiple actions of PEDF are likely conserved in evolution since it has the same gene structure across phyla, although the size of the gene ranges from 48.3 kb in X. tropicalis to 2.9 kb in fugu, with human PEDF at a size of 15.6 kb. A strong similarity in the proximal 200 bp of the PEDF promoter in mammals suggests the existence of a possible regulatory region across phyla. Using a non-synonymous/synonymous substitution rate ratio we show that mammalian and fish PEDFs have similar ratios of <0.13, reflecting a strong purifying selection of PEDF gene. A large number of repetitive transposable elements of the SINE and LINE class were found with random distribution in both the promoter and introns of mammalian PEDF. CONCLUSION: The PEDF gene first appears in vertebrates and our studies suggest that the regulation and biological actions of this gene are preserved across vertebrates. This comprehensive analysis of the PEDF gene across phyla provides new information that will aid further characterization of common functional motifs of this serpin in biological processes.

Animals↗
PubMed2006

PEDF induces apoptosis in human endothelial cells by activating p38 MAP kinase dependent cleavage of multiple caspases.

We examined how pigment epithelium derived factor (PEDF), an effective endogenous antiangiogenic protein, decreases survival of primary cultures of human umbilical vein endothelial cells (HUVECs) in a low serum environment supplemented with the endothelial cell growth factor (VEGF). We provide evidence that induction of apoptosis by PEDF is associated with activation of p38 followed by cleavage of caspases 3, 8, and 9 by treatment with PEDF, and PEDF's actions are caspase dependent. A key mediator in the executioner effects of PEDF is p38 since the inhibition of p38 activity blocked apoptosis and prevented cleavage of caspases 3, 8, and 9. Although PEDF-induced phosphorylation of JNK1, the inhibition of JNK1 had no effect on apoptosis, even though it prevented phosphorylation of JNK1 by PEDF. Based on these findings, we propose that the antiangiogenic action of PEDF is dependent on activation of p38 MAPkinase which regulates cleavage of multiple caspases cascades.

Apoptosis↗
PubMed2006

A PEDF N-terminal peptide protects the retina from ischemic injury when delivered in PLGA nanospheres.

The neuroprotective effects of small pigment epithelium-derived factor (PEDF) peptides injected intravitreally as free peptides or delivered in poly(lactide-co-glycolide) (PLGA) nanospheres, were tested in retinal ischemic injury. We induced transient ischemia in C57BL/6 mice by elevating the intraocular pressure to the equivalent of 120 mmHg for 60 min, then injected these eyes with one of the following: PBS, full-length native PEDF, N-terminal peptides-PEDF(136-155) and PEDF(82-121), blank PLGA nanospheres or PLGA loaded with PEDF(82-121) (PLGA-PEDF(82-121)). Morphometric analysis and TUNEL assays were used to determine the extent of retinal damage. Transient ischemia caused a rapid reduction in the number of viable cells in the retinal ganglion cell (RGC) layer over 48h as compared to non-ischemic retinas. About 76% surviving cells in the RGC layer were observed in the full-length PEDF protein treated group, whereas only 32% of cells survived in the PBS group. Thus, PEDF prevented approximately 44% of the cell death in the RGC layer resulting from transient ischemia. PEDF(82-121) peptide was as effective as full-length PEDF when injected as either a free peptide or delivered in PLGA nanospheres. PLGA-PEDF(82-121) showed longer-term protection of the RGC layer with no noticeable side effects at 7days. PEDF and PEDF(82-121) lessened damage to the IPL as measured by layer thickness. PEDF and PEDF(82-121) also delayed retinal responses to ischemic injury as measured by GFAP immunolabeling in Müller cells. PEDF(82-121) is an effective neuroprotective peptide in retinal ischemia. PLGA-PEDF(82-121) offers greater protection to the retina suggesting that this peptide and the method of delivering therapeutically active drugs have potential clinical advantages for longer-term treatments of retinal diseases.

Animals↗
PubMed2005

Comprehensive in silico functional specification of mouse retina transcripts.

BACKGROUND: The retina is a well-defined portion of the central nervous system (CNS) that has been used as a model for CNS development and function studies. The full specification of transcripts in an individual tissue or cell type, like retina, can greatly aid the understanding of the control of cell differentiation and cell function. In this study, we have integrated computational bioinformatics and microarray experimental approaches to classify the tissue specificity and developmental distribution of mouse retina transcripts. RESULTS: We have classified a set of retina-specific genes using sequence-based screening integrated with computational and retina tissue-specific microarray approaches. 33,737 non-redundant sequences were identified as retina transcript clusters (RTCs) from more than 81,000 mouse retina ESTs. We estimate that about 19,000 to 20,000 genes might express in mouse retina from embryonic to adult stages. 39.1% of the RTCs are not covered by 60,770 RIKEN full-length cDNAs. Through comparison with 2 million mouse ESTs, spectra of neural, retinal, late-generated retinal, and photoreceptor -enriched RTCs have been generated. More than 70% of these RTCs have data from biological experiments confirming their tissue-specific expression pattern. The highest-grade retina-enriched pool covered almost all the known genes encoding proteins involved in photo-transduction. CONCLUSION: This study provides a comprehensive mouse retina transcript profile for further gene discovery in retina and suggests that tissue-specific transcripts contribute substantially to the whole transcriptome.

Animals↗
PubMed2005

Uncoupling protein-2 is critical for nigral dopamine cell survival in a mouse model of Parkinson's disease.

Mitochondrial uncoupling proteins dissociate ATP synthesis from oxygen consumption in mitochondria and suppress free-radical production. We show that genetic manipulation of uncoupling protein-2 (UCP2) directly affects substantia nigra dopamine cell function. Overexpression of UCP2 increases mitochondrial uncoupling, whereas deletion of UCP2 reduces uncoupling in the substantia nigra-ventral tegmental area. Overexpression of UCP2 decreased reactive oxygen species (ROS) production, which was measured using dihydroethidium because it is specifically oxidized to fluorescent ethidium by the superoxide anion, whereas mice lacking UCP2 exhibited increased ROS relative to wild-type controls. Unbiased electron microscopic analysis revealed that the elevation of in situ mitochondrial ROS production in UCP2 knock-out mice was inversely correlated with mitochondria number in dopamine neurons. Lack of UCP2 increased the sensitivity of dopamine neurons to 1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine (MPTP), whereas UCP2 overexpression decreased MPTP-induced nigral dopamine cell loss. The present results expose the critical importance of UCP2 in normal nigral dopamine cell metabolism and offer a novel therapeutic target, UCP2, for the prevention/treatment of Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2005

STAT3 activation in response to growth factors or cytokines participates in retina precursor proliferation.

Growth factors and cytokines play an important role in the development of central nervous systems including neurons of the retina. However, the molecular pathways that trigger cell growth remain unclear in neuronal precursors. In the present studies, we used a retinal explant culture system to investigate the response of signal transducer and activator of transcription factors (STATs) to extrinsic factors during mouse retinal development. Retinas from embryonic and neonatal stages showed that STAT3 but not STAT1 was activated in response to ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), fibroblast growth factor-1 (FGF1), fibroblast growth factor-2 (FGF2), epidermal growth factor (EGF), interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) in distinct patterns. STAT3 activation was detected in the outermost retina layer in response to CNTF, LIF, FGF1, and IFN-alpha 24 hr after stimulation in postnatal day 1 (PN1) explants, but not FGF2, EGF, IFN-gamma, and retinoic acid (RA). Cytokine stimulation increased the number of cells incorporating BrdU and the labelled cells co-localized with phosphorylated STAT3, indicating that STAT3 may play an essential role in coupling extrinsic factors to retina precursor cell (RPC) proliferation. Furthermore, persistent expression of two neural precursor markers, Hes1 and Otx2 was detected in outer retinal layers and correlated with STAT3 activation by CNTF, suggesting that STAT3 activation may play a critical role in stimulating mitotic precursors. These results strongly support a model that STAT3-mediated signalling regulates precursor populations during mouse retina development.

Animals↗
PubMed2005

PEDF and the serpins: phylogeny, sequence conservation, and functional domains.

Pigment epithelium derived factor (PEDF) is non-inhibitory serpin with neurotrophic and antiangiogenic functions. In this study, we have assembled PEDF sequences for 9 additional species by data base mining and performed cross-species alignment for 14 PEDF sequences to identify conserved structural domains. We found evolutionary conservation of a leader sequence, a single C-terminal glycosylation site, collagen-binding residues, and four specific conserved PEDF peptides. The C-terminus, 384--415 and an N-terminal region 78--95, show close homology with many other serpins, and there is strong conservation of 39 of 51 consensus key residues involved in serpin structure and function. Two peptide regions, 40--67 and 277--301, are unique to PEDF but conserved in all species. Conserved residues at the N-terminus, helix d (hD), and helix A (hA) of PEDF form a structure similar to the heparin-binding groove of other serpins. We identified a motif in PEDF that is homologous to the nuclear localization signals of other proteins. A bitopographical localization of PEDF was confirmed by immunocytochemistry and Western blots. Our results suggest that secretion is required for PEDF's activity, that PEDF can migrate to the nucleus, and that PEDF has structural and functional features more common with inhibitory serpins.

Amino Acid Motifs↗
PubMed2004

Modulation of synaptic function by cGMP and cGMP-gated cation channels.

Cyclic nucleotide-gated cation channels have been studied intensively in the primary sensory neurons of the visual and olfactory systems. Using both anatomical and physiological methods we have shown that they have a much more widespread distribution in the nervous system. In many retinal ganglion cells cGMP, but not cAMP, activates a non-selective conductance that has many of the properties of CNG channels. As many neurons also contain cGMP-dependent protein kinases (PKGs), we have used a variety of cGMP analogues to distinguish the actions of cGMP. Sp-8-Br-PET-cGMPS is a potent non-hydrolyzable cGMP analogue that is an agonist of PKG. We found that Sp-8-Br-PET-cGMPS acts as a competitive inhibitor of at least the rod CNG channel. Rp-8-Br-cGMPS has shown the opposite effects, namely as an agonist of the rod CNG channel and an inhibitor of PKG. In dissociated cell cultures and slices of rodent visual cortex cGMP had multiple rapid and reversible effects on transmission at glutamatergic synapses. Extracellular application of 8-Br-cGMP or Sp-8-Br-PET-cGMPS reduced stimulus evoked EPSPs in cortical slices. In cortical cultures both analogs reduced the frequency of spontaneous EPSCs, but not their amplitude. The effects on both EPSPs and EPSCs were presynaptic. The effects on evoked EPSPs may be due, in part, to reduced calcium influx through voltage-gated calcium channels. The effects on spontaneous EPSCs may be due, in part, to modulation of calcium fluxes through internal stores. Similar modulations of synaptic transmission have been found at gabaergic synapses. On postsynaptic cells, PKG activation produced a dramatic enhancement of the responses to applied NMDA. No effects were detected on applied AMPA/kainate or GABA. Together the results suggest that cGMP may use multiple mechanisms to modulate synaptic efficacy and that its actions may include regulating synaptic plasticity and the relative strength of excitatory and inhibitory drive through neural pathways.

Animals↗
PubMed2004

Neuroprotective and antiangiogenic actions of PEDF in the eye: molecular targets and therapeutic potential.

Pigment epithelium-derived factor (PEDF) is a 50-kDa protein encoded by a single gene that shows strong conservation across phyla from fish to mammals. It is secreted by the retinal pigment epithelium (RPE) and a select number of other cell types in the eye, as well as by other tissues in the body. PEDF was originally defined by its ability to induce differentiation in retinoblastoma cells. It also promotes a non-proliferative, differentiated state in a number of other cell types. PEDF protects retinal neurons from light damage, oxidative stress and glutamate excitotoxicity. PEDF is also antiangiogenic and can inhibit the growth of blood vessels in the eye induced in a variety of ways. A balance in the levels of PEDF and the proangiogenic factor vascular endothelial growth factor-A is perturbed in a range of retinal neovascular diseases. Some of the pathways by which PEDF exerts its actions on cells have now been defined. Peptide fragments of PEDF carry biological activity and may be valuable therapeutic agents that readily penetrate the eye.

Amino Acid Sequence↗
PubMed2004

STAT3-mediated signaling in the determination of rod photoreceptor cell fate in mouse retina.

PURPOSE: The purpose of this study was to determine the intracellular pathways by which ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) negatively regulate the development of rod photoreceptors in the mouse retina. METHODS: Retina explant cultures derived from timed-pregnant CD-1 mice were used to monitor rod photoreceptor differentiation. CNTF was used to activate the signal transducer and activator of transcription (STAT)-3 and mitogen-activated protein kinase (MAPK) signal transduction pathways. Activation of STAT3 and MAPK were manipulated by using dominant-negative STAT3 recombinant adenoviruses and a specific inhibitor of MAPK, respectively. Explanted retinas were harvested at distinct time points and processed for immunohistochemistry. RESULTS: Blocking of the MAPK pathway by the MAPK inhibitor PD98059 did not affect normal development of rods in retina explants or the suppression of their appearance by treatment with CNTF. In contrast, activated STAT3 was necessary for suppression of the rod cell fate decision. A deficiency of the STAT3 pathway induced by a dominant negative STAT3 abolished inhibition of rod development by CNTF. CONCLUSIONS: These results indicate that STAT3, but not MAPK, can critically regulate photoreceptor development during mouse retina development.

Animals↗
PubMed2003

Uncoupling protein 2 prevents neuronal death including that occurring during seizures: a mechanism for preconditioning.

The mitochondrial uncoupling protein (UCP2) is expressed in selected regions of the brain. Here we demonstrate that up-regulation of UCP2 is part of a neuroprotective set of responses to various cellular stresses in vitro and in vivo. PC12 cells, when transfected with UCP2, were protected against free radical-induced cell death. Seizure activity was associated with elevated UCP2 levels and mitochondrial uncoupling activity. In transgenic mice that expressed UCP2 constitutively in the hippocampus before seizure induction, a robust reduction in cell death was seen. Because UCP2 increased mitochondrial number and ATP levels with a parallel decrease in free radical-induced damage, it is reasonable to suggest that mitochondrial UCPs precondition neurons by dissociating cellular energy production from that of free radicals to withstand the harmful effects of cellular stress occurring in a variety of neurodegenerative disorders, including epilepsy.

Adenosine Diphosphate↗
PubMed2003

Developmental and tissue expression patterns of mouse Mpp4 gene.

The temporal and spatial expression patterns of mouse membrane palmitoylated protein 4 (Mpp4) gene was investigated. Mpp4 was expressed in postnatal but not embryonic retina by microarray analysis. Real-time quantitative RT-PCR analysis showed that, in addition to retina, Mpp4 was expressed at much lower levels in brain, heart, liver, and spleen tissues. In situ hybridization revealed that Mpp4 was exclusively localized in the photoreceptor cells. It was also detected in pineal gland but not other regions of the brain. Immunofluorescence labeling on eye sections of wild-type mice and transgenic mice with cone-specific GFP expression demonstrated that Mpp4 protein was localized at rod but not at cone photoreceptor synaptic terminals. The high level and cell-type specific expression of mouse Mpp4 gene makes it a good candidate for the targeting and assembly of specific molecules, such as calcium channels, at rod synaptic terminals.

Animals↗
PubMed2003

Expression and activation of STAT proteins during mouse retina development.

Cytokines and growth factors play important roles in mammalian ocular development and maintenance. Recent studies have indicated that some of these ligands can activate signal transducer and activator of transcription factors (STATs) and modulate gene transcription. The purpose of this study was to investigate the expression and activation of STAT proteins in the developing mouse retina. Anti-STAT and anti-phosphorylated STAT antibodies were used to detect the expression and activation of STATs in embryonic and postnatal neuronal retina, ciliary margin, and retinal pigment epithelium (RPE). In situ hybridization and Western blot were also employed. In embryonic stages, all STAT proteins were expressed in the neuronal retina in distinct cell populations at different embryonic stages. For example, Stat3 expression and activation gradually increased in the inner neuroblast layer and ciliary margin during development. In adult retina, Stat3 was detected in the inner nuclear layer and ganglion cells layers. Stat1 was strongly expressed in both outer and inner plexiform layers. Stat5a was clearly expressed in the outer/inner nuclear layer, the ganglion cell layer, and the inner plexiform layer. Strong expression of Stat3, Stat5a, and Stat6 was observed in the RPE. Activated Stat3 and Stat5a were found in the neural retina and the RPE. Distinct STAT proteins were present in different cell populations in neuronal retina and RPE suggesting multiple functions of STATs in mammalian eye development. Studies of STAT signal pathways in the eye may contribute to the understanding of molecular mechanisms in control of ocular development and pathogenesis.

Animals↗
PubMed2003

Therapeutic prospects for PEDF: more than a promising angiogenesis inhibitor.

Blood vessel growth and stability are under the exquisite control of a network of pro- and anti-angiogenic factors. Disruption of the balance between these factors is a characteristic of tumor growth and many vascular diseases. Endogenous angiogenesis inhibitors, particularly those that act broadly at the earliest stages, could be excellent pharmacological tools in combating pathogenic vessel growth. Pigment-epithelium-derived factor (PEDF) is a natural angiogenesis inhibitor that (1) targets only new vessel growth, (2) can be administered therapeutically as a soluble protein or by viral-mediated gene transfer, (3) is stable and nontoxic when injected, and (4) is more potent than other well-characterized angiogenesis inhibitors. Because PEDF also has differentiating and neuroprotective activities, it has additional benefits for use in the nervous system. The production of PEDF by many tissues suggests its therapeutic potential should be explored in a much wider range of diseases, including tumor proliferation and metastasis.

Angiogenesis Inducing Agents↗
PubMed2003

Coenzyme Q induces nigral mitochondrial uncoupling and prevents dopamine cell loss in a primate model of Parkinson's disease.

Parkinson's disease is characterized by dopamine cell loss of the substantia nigra. Parkinson's disease and the neurotoxin 1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine may destroy dopamine neurons through oxidative stress. Coenzyme Q is a cofactor of mitochondrial uncoupling proteins that enhances state-4 respiration and eliminate superoxides. Here we report that short-term oral administration of coenzyme Q induces nigral mitochondrial uncoupling and prevents dopamine cell loss after 1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine administration in monkeys.

Animals↗