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PubMed2005

Generation of Rx+/Pax6+ neural retinal precursors from embryonic stem cells.

We report directed differentiaion of retinal precursors in vitro from mouse ES cells. Six3+ rostral brain progenitors are generated by culturing ES cells under serum-free suspension conditions (SFEB culture) in the presence of Wnt and Nodal antagonists (Dkk1 and LeftyA), and subsequently steered to differentiate into Rx+ cells (16%) by treatment with activin and serum. Consistent with the characteristics of early neural retinal precursors, the induced Rx+ cells coexpress Pax6 and the mitotic marker Ki67, but not Nestin. The ES cell-derived precursors efficiently generate cells with the photoreceptor phenotype (rhodopsin+, recoverin+) when cocultured with embryonic retinal cells. Furthermore, organotypic culture studies demonstrate the selective integration and survival of ES cell-derived cells with the photoreceptor phenotype (marker expression and morphology) in the outer nuclear layer of the retina. Taken together, ES cells treated with SFEB/Dkk1/LeftyA/serum/activin generate neural retinal precursors, which have the competence of photoreceptor differentiation.

Activins↗
PubMed1996

Inhibition of G protein-coupled receptor kinase subtypes by Ca2+/calmodulin.

G protein-coupled receptor kinases (GRKs) are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6. The functional state of the GRKs can be actively regulated in different ways. In particular, it was found that retinal rhodopsin kinase (GRK1), but not the ubiquitous betaARK1 (GRK2), can be inhibited by the photoreceptor-specific Ca2+-binding protein recoverin through direct binding. The present study was aimed to investigate regulation of other GRKs by alternative Ca2+-binding proteins such as calmodulin (CaM). We found that Gbetagamma-activated GRK2 and GRK3 were inhibited by CaM to similar extents (IC50 approximately 2 microM), while a 50-fold more potent inhibitory effect was observed on GRK5 (IC50 = 40 nM). Inhibition by CaM was strictly dependent on Ca2+ and was prevented by the CaM inhibitor CaMBd. Since Gbetagamma, which is a binding target of Ca2+/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is Gbetagamma-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduced GRK5 binding to the membrane. The mechanism of GRK5 inhibition appeared to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases.

Calcium↗
PubMed1997

Identification and quantitation of the fatty acids composing the CoA ester pool of bovine retina, heart, and liver.

Several proteins found in retinal photoreceptor cells (guanylate cyclase activating protein, protein kinase A, recoverin, and transducin) are N-terminally modified with the fatty acids 12:0, 14:0, 14:1n-9, and 14:2n-6, whereas similar proteins in other tissues contain only 14:0. It has been hypothesized that the acyl-CoA pool of the retina contains amounts of 12:0, 14:1n-9, and 14:2n-6 elevated over 14:0, in comparison to other tissues, and this accounts for the specificity of N-terminal fatty acylation. To test this hypothesis, we performed fatty acid analysis on total acyl-CoAs purified from bovine retina (light-adapted), heart, and liver. We also examined the N- and S-linked fatty acid composition of the total protein pools from these tissues. Acyl-CoAs were prepared from heart, liver, and retina and separated by high performance liquid chromatography (HPLC). Identities of peaks were based on HPLC of standard 12:0, 14:0, 14:1n-9, and 14:2n-6 CoAs. Total protein was subjected to base hydrolysis followed by acidic methanolysis to release S- and N-linked fatty acids, respectively, and fatty acid phenacyl esters were prepared for HPLC analysis. Retina had levels of 12:0 (2.7 +/- 2.1%), 14:1n-9 (2.9 +/- 2.2%), and 14:2n-6 (1.6 +/- 0.7%) CoAs below that of 14:0 CoA (7.0 +/- 1.8%). Likewise, heart levels of 14:2n-6 CoA (3.7 +/- 0.1%) were near and 12:0 (2.6 +/- 0. 6%) and 14:1n-9 (0.7 +/- 0.3%) CoAs were below that of 14:0 CoA (3.8 +/- 1.0%). Liver had levels of 12:0 (16.1 +/- 5.7%) and 14:2n-6 (8.1 +/- 1.2%) CoAs above and 14:1n-9 CoA (1.2 +/- 0.6%) below that of 14:0 CoA (5.9 +/- 0.8%). Fatty acid analysis of total protein showed that all tissues contained S-linked 16:0, 18:0, and 18:1n-9. Retina proteins contained N-linked 14:0, 14:1n-9, and 14:2n-6, whereas heart and liver had only 14:0. Our findings do not support the hypothesis that the CoA ester pool of the retina is enriched with 12:0, 14:1n-9, and 14:2n-6 over 14:0, in comparison to other tissues. This suggests that alternative models must be considered for the regulation of N-terminal fatty acylation of proteins in photoreceptor cells.

Acyl Coenzyme A↗
PubMed1999

Neuronal Ca(2+) sensor 1. Characterization of the myristoylated protein, its cellular effects in permeabilized adrenal chromaffin cells, Ca(2+)-independent membrane association, and interaction with binding proteins, suggesting a role in rapid Ca(2+) signal transduction.

Overexpression of frequenin and its orthologue neuronal Ca(2+) sensor 1 (NCS-1) has been shown to increase evoked exocytosis in neurons and neuroendocrine cells. The site of action of NCS-1 and its biochemical targets that affect exocytosis are unknown. To allow further investigation of NCS-1 function, we have demonstrated that NCS-1 is a substrate for N-myristoyltransferase and generated recombinant myristoylated NCS-1. The bacterially expressed NCS-1 shows Ca(2+)-induced conformational changes. The possibility that NCS-1 directly interacts with the exocytotic machinery to enhance exocytosis was tested using digitonin-permeabilized chromaffin cells. Exogenous NCS-1 was retained in permeabilized cells but had no effect on Ca(2+)-dependent release of catecholamine. In addition, exogenous NCS-1 did not regulate cyclic nucleotide levels in this system. These data suggest that the effects of NCS-1 seen in intact cells are likely to be due to an action on the early steps of stimulus-secretion coupling or on Ca(2+) homeostasis. Myristoylated NCS-1 bound to membranes in the absence of Ca(2+) and endogenous NCS-1 was tightly membrane-associated. Using biotinylated NCS-1, a series of specific binding proteins were detected in cytosol, chromaffin granule membrane, and microsome fractions of adrenal medulla. These included proteins distinct from those detected by biotinylated calmodulin, demonstrating the presence of multiple specific Ca(2+)-independent and Ca(2+)-dependent binding proteins as putative targets for NCS-1 action. A model for NCS-1 function, from these data, indicates a constitutive membrane association independent of Ca(2+). This differs from the Ca(2+) myristoyl switch model for the closely related recoverin and suggests a possible action in rapid Ca(2+) signal transduction in response to local Ca(2+) signals.

Adrenal Medulla↗
PubMed2000

Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin.

Five members of a novel Ca(2+)-binding protein subfamily (CaBP), with 46-58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca(2+)-binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca(2+) coordination and the fact that their central alpha-helixes are extended by one alpha-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca(2+)-mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.

Amino Acid Sequence↗
PubMed2001

Calsenilin is a substrate for caspase-3 that preferentially interacts with the familial Alzheimer's disease-associated C-terminal fragment of presenilin 2.

Calsenilin is a member of the recoverin family of neuronal calcium-binding proteins that we have previously shown to interact with presenilin 1 (PS1) and presenilin 2 (PS2) holoproteins. The expression of calsenilin can regulate the levels of a proteolytic product of PS2 (Buxbaum, J. D., Choi, E. K., Luo, Y., Lilliehook, C., Crowley, A. C., Merriam, D. E., and Wasco, W. (1998) Nat. Med. 4, 1177-1181) and reverse the presenilin-mediated enhancement of calcium signaling (Leissring, M. A., Yamasaki, T. R., Wasco, W., Buxbaum, J. D., Parker, I., and LaFerla, F. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8590-8593). Here, we have used cultured mammalian cells that transiently or stably express calsenilin to extend the characterization of calsenilin and of the calsenilin-PS2 interaction. We have found that calsenilin has the ability to interact with endogenous 25-kDa C-terminal fragment (CTF) that is a product of regulated endoproteolytic cleavage of PS2 and that the presence of the N141I PS2 mutation does not significantly alter the interaction of calsenilin with PS2. Interestingly, when the 25-kDa PS2 CTF and the 20-kDa PS2 CTF are both present, calsenilin preferentially interacts with the 20-kDa CTF. Increases in the 20-kDa fragment are associated with the presence of familial Alzheimer's disease-associated mutations (Kim, T., Pettingell, W. H., Jung, Y., Kovacs, D. M., and Tanzi, R. E. (1997) Science 277, 373-376). However, the finding that the production of the 20-kDa fragment is regulated by the phosphorylation of PS2 (Walter, J., Schindzielorz, A., Grunberg, J., and Haass, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1391-1396) suggests that it is a regulated physiological event that also occurs in the absence of the familial Alzheimer's disease-associated mutations in PS2. Finally, we have demonstrated that calsenilin is a substrate for caspase-3, and we have used site-directed mutagenesis to map the caspase-3 cleavage site to a region that is proximal to the calcium binding domain of calsenilin.

Alzheimer Disease↗
PubMed2001

Calcium-regulated DNA binding and oligomerization of the neuronal calcium-sensing protein, calsenilin/DREAM/KChIP3.

Calsenilin/DREAM/KChIP3, a member of the recoverin branch of the EF-hand superfamily, interacts with presenilins, serves as a calcium-regulated transcriptional repressor, and interacts with A-type potassium channels. Here we report physicochemical characterization of calcium binding, oligomerization, and DNA binding of human calsenilin/DREAM/KChIP3. Equilibrium Ca(2+) binding measurements indicate that the protein binds 3 Ca(2+) with a dissociation constant of 14 microM and a Hill coefficient of 0.7. Dynamic light scattering and size exclusion chromatography show that the Ca(2+)-bound protein exists as a dimer at protein concentrations lower than 150 microM and forms a tetramer at concentrations above 200 microM. The Ca(2+)-free protein is a tetramer in the concentration range 20-450 microM. Isothermal titration calorimetry and dynamic light scattering indicate that the Ca(2+)-free protein tetramer binds endothermically (DeltaH = +25 kcal/mol) to four molecules of DNA derived from the downstream regulatory element (DRE) of either the prodynorphin or c-fos genes. One DRE molecule binds tightly to the protein with a dissociation constant (K(d)) of 75 nM, and the other three bind more weakly (K(d) = 640 nM). No significant DNA binding was observed for the Ca(2+)-bound protein. The N-terminal protein fragment (residues 1-70) binds nonspecifically to DRE in a Ca(2+)-independent manner, whereas a C-terminal fragment containing the four EF-hands (residues 65-256) binds DRE (K(d) = 200 nM) in a Ca(2+)-regulated and sequence-specific fashion. The C-terminal fragment is a tetramer in the Ca(2+)-free state and dissociates into dimers at saturating Ca(2+) levels.

Amino Acid Sequence↗
PubMed2001

Instead of binding calcium, one of the EF-hand structures in guanylyl cyclase activating protein-2 is required for targeting photoreceptor guanylyl cyclase.

Guanylyl cyclase activator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca(2+)-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca(2+) concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca(2+) and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca(2+)-sensitive dimerization. Point mutations within the EF-1 region can interfere with both the activation of the cyclase by the Ca(2+)-free form of GCAP-2 and the inhibition of retGC basal activity by the Ca(2+)-loaded GCAP-2. Our results strongly indicate that evolutionary conserved and GCAP-specific amino acid residues within the EF-1 can create a contact surface for binding GCAP-2 to the cyclase. Apparently, in the course of evolution GCAP-2 exchanged the ability of its first EF-hand motif to bind Ca(2+) for the ability to interact with the target enzyme.

Amino Acid Sequence↗
PubMed2002

Modulation of Kv4-encoded K(+) currents in the mammalian myocardium by neuronal calcium sensor-1.

Voltage-gated K(+) channels are multimeric proteins, consisting of four pore-forming alpha-subunits alone or in association with accessory subunits. Recently, for example, it was shown that the accessory Kv channel interacting proteins form complexes with Kv4 alpha-subunits and modulate Kv4 channel activity. The experiments reported here demonstrate that the neuronal calcium sensor protein-1 (NCS-1), another member of the recoverin-neuronal calcium sensor superfamily, is expressed in adult mouse ventricles and that NCS-1 co-immunoprecipitates with Kv4.3 from (adult mouse) ventricular extracts. In addition, co-expression studies in HEK-293 cells reveal that NCS-1 increases membrane expression of Kv4 alpha-subunits and functional Kv4-encoded K(+) current densities. Co-expression of NCS-1 also decreases the rate of inactivation of Kv4 alpha-subunit-encoded K(+) currents. In contrast to the pronounced effects of Kv channel interacting proteins on Kv4 channel gating, however, NCS-1 co-expression does not measurably affect the voltage dependence of steady-state inactivation or the rate of recovery from inactivation of Kv4-encoded K(+) currents. Taken together, these results suggest that NCS-1 is an accessory subunit of Kv4-encoded I(to,f) channels that functions to regulate I(to,f) density in the mammalian myocardium.

Adenoviridae↗
PubMed2002

Phosphatidylinositol 4-OH kinase is a downstream target of neuronal calcium sensor-1 in enhancing exocytosis in neuroendocrine cells.

Neuronal calcium sensor-1 (NCS-1), the mammalian orthologue of frequenin, belongs to a family of EF-hand-containing Ca(2+) sensors. NCS-1/frequenin has been shown to enhance synaptic transmission in PC12 cells and Drosophila and Xenopus, respectively. However, the precise molecular mechanism for the enhancement of exocytosis is largely unknown. In PC12 cells, NCS-1 potentiated exocytosis evoked by ATP, an agonist to phospholipase C-linked receptors, but had no effect on depolarization-evoked release. NCS-1 also enhanced exocytosis triggered by ionomycin, a Ca(2+) ionophore that bypasses K(+) and Ca(2+) channels. Overexpression of NCS-1 caused a shift in the dose-response curve of inhibition of ATP-evoked secretion using phenylarsine oxide, an inhibitor of phosphatidylinositol 4-OH kinase (PI4K). Plasma membrane phosphatidylinositol 4,5-bisphosphate pools were increased upon NCS-1 transfection as visualized using a phospholipase C-delta pleckstrin homology domain-green fluorescent protein construct. NCS-1-transfected cell extracts displayed increased phosphatidylinositol-4-phosphate biosynthesis, indicating an increase in PI4K activity. Mutations in NCS-1 equivalent to those that abolish the interaction of recoverin, another EF-hand-containing Ca(2+) sensor, with its downstream target rhodopsin kinase, lost their ability to enhance exocytosis. Taken together, the present data indicate that NCS-1 modulates the activity of PI4K, leading to increased levels of phosphoinositides and concomitant enhancement of exocytosis.

1-Phosphatidylinositol 4-Kinase↗
PubMed2004

Identification of residues that determine the absence of a Ca(2+)/myristoyl switch in neuronal calcium sensor-1.

The neuronal calcium sensor (NCS) family of Ca(2+)-binding proteins regulates a number of different processes in neurons and photoreceptor cells. The first of these proteins to be characterized, recoverin, was shown to exhibit a Ca(2+)/myristoyl switch whereby its N-terminal myristoyl group is sequestered in the Ca(2+)-free form and is exposed on Ca(2+) binding to allow the protein to become membrane-associated. It has subsequently been shown that certain other family members also exhibit this mechanism in living cells. In contrast, NCS-1 does not show the Ca(2+)/myristoyl switch and is membrane-associated even at low Ca(2+) concentrations. We have used sequence comparison combined with information from structural analyses to attempt to identify candidate residues within the NCS proteins that determine whether or not the Ca(2+)/myristoyl switch operates in cells and have tested their functional significance by mutagenesis. The results show that NCS-1 possesses residues within its N terminus that lock the myristoyl group in an exposed conformation. In addition, other structural aspects within the C-terminal domains are required to allow the switch to operate. We have determined a key role for residues within the motif EELTRK in NCS-1 in keeping the myristoyl group exposed and allowing the protein to be constitutively membrane-associated.

Amino Acid Sequence↗
PubMed1996

The kinetics of inactivation of the rod phototransduction cascade with constant Ca2+i.

A rich variety of mechanisms govern the inactivation of the rod phototransduction cascade. These include rhodopsin phosphorylation and subsequent binding of arrestin; modulation of rhodopsin kinase by S-modulin (recoverin); regulation of G-protein and phosphodiesterase inactivation by GTPase-activating factors; and modulation of guanylyl cyclase by a high-affinity Ca(2+)-binding protein. The dependence of several of the inactivation mechanisms on Ca2+i makes it difficult to assess the contributions of these mechanisms to the recovery kinetics in situ, where Ca2+i is dynamically modulated during the photoresponse. We recorded the circulating currents of salamander rods, the inner segments of which are held in suction electrodes in Ringer's solution. We characterized the response kinetics to flashes under two conditions: when the outer segments are in Ringer's solution, and when they are in low-Ca2+ choline solutions, which we show clamp Ca2+i very near its resting level. At T = 20-22 degrees C, the recovery phases of responses to saturating flashes producing 10(2.5)-10(4.5) photoisomerizations under both conditions are characterized by a dominant time constant, tau c = 2.4 +/- 0.4 s, the value of which is not dependent on the solution bathing the outer segment and therefore not dependent on Ca2+i. We extended a successful model of activation by incorporating into it a first-order inactivation of R*, and a first-order, simultaneous inactivation of G-protein (G*) and phosphodiesterase (PDE*). We demonstrated that the inactivation kinetics of families of responses obtained with Ca2+i clamped to rest are well characterized by this model, having one of the two inactivation time constants (tau r* or tau PDE*) equal to tau c, and the other time constant equal to 0.4 +/- 0.06 s.

Animals↗
PubMed1999

Flagellar protein localization mediated by a calcium-myristoyl/palmitoyl switch mechanism.

The mechanisms by which proteins are targeted to flagella and cilia are poorly understood. We set out to determine the basis for the specific localization of a 24 kDa flagellar calcium-binding protein (FCaBP) expressed in all life cycle stages of Trypanosoma cruzi. Through the study of trypanosome transfectants expressing various FCaBP deletion mutants, we found that the N-terminal 24 amino acids of the protein are necessary and sufficient for flagellar localization. Subsequent experiments revealed that FCaBP is myristoylated and palmitoylated and, in fact, is one of very few proteins in the cell possessing these acyl modifications. Both fatty acids are required for flagellar localization, suggesting that FCaBP localization may be mediated through association with the flagellar plasma membrane. Indeed, FCaBP associates with the flagellar membrane in a calcium-dependent manner, reminiscent of the recoverin family of calcium-myristoyl switch proteins. Thus, FCaBP is a novel member of the calcium-acyl switch protein family and is the only member described to date that requires two fatty acid modifications for specific membrane association. Its unique localization mechanism is the first described for any flagellar protein. The existence of such a protein in this protozoan suggests that acylation and calcium switch mechanisms for regulated membrane association are conserved among eukaryotes.

Amino Acid Sequence↗
PubMed1998

Ocular manifestations of cancer.

Cancer may affect the eye and orbit by a direct effect of metastatic neoplastic infiltration or compression or by circulating antibodies involving paraneoplastic retinal degeneration. Metastatic tumor to the uvea is the most common form of an intraocular malignant process. The choroid is the most common site for uveal metastasis; metastases to the ciliary body, iris, retina, optic disc, and vitreous are rare. Approximately one third of patients have no history of primary cancer at the time of ocular diagnosis. Breast and lung carcinomas for women and lung and gastrointestinal carcinomas for men most commonly metastasize to the eye and orbit. The short-term prognosis for vision is usually good but the systemic prognosis is poor. The visual paraneoplastic syndromes encompass several distinct clinical and pathologic entities including carcinoma-associated retinopathy, melanoma-associated retinopathy, and bilateral diffuse melanocytic uveal proliferation. The first affects photoreceptors, the second is thought to affect bipolar cell function, and the third targets the uveal tract. Identification of circulating antibodies against retinal proteins (recoverin, 23-kD retinal protein; 46-kD and 60-kD retinal proteins) serves to recognize the paraneoplastic nature of the patient's symptoms, which frequently develop before the cancer is diagnosed. No therapy exists to stop the inexorable progressive loss of vision. Metastasis to the eye and orbit and paraneoplastic disorders represent a very bad prognostic sign. Recognition of their visual symptoms and ocular findings should alert the ophthalmologist to the possibility of cancer and systemic evaluation should be pursued.

Eye Neoplasms↗
PubMed1997

Anatomical evidence for rod input to the parvocellular pathway in the visual system of the primate.

The question whether midget bipolar cells in macaque monkey retina receive input from rods was investigated using double-label immunocytochemistry. Flat midget bipolar cells (labelled with antibodies against recoverin) were found to be pre- and postsynaptic to All amacrine cells (labelled with antibodies against calretinin). These results support physiological evidence that rod photoreceptor signals could reach the parvocellular pathway at an early stage of visual processing.

Animals↗
PubMed1998

G protein-coupled receptor kinases.

G protein-coupled receptor kinases (GRKs) constitute a family of six mammalian serine/threonine protein kinases that phosphorylate agonist-bound, or activated, G protein-coupled receptors (GPCRs) as their primary substrates. GRK-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling, or desensitization. This review focuses on the regulation of GRK activity by a variety of allosteric and other factors: agonist-stimulated GPCRs, beta gamma subunits of heterotrimeric GTP-binding proteins, phospholipid cofactors, the calcium-binding proteins calmodulin and recoverin, posttranslational isoprenylation and palmitoylation, autophosphorylation, and protein kinase C-mediated GRK phosphorylation. Studies employing recombinant, purified proteins, cell culture, and transgenic animal models attest to the general importance of GRKs in regulating a vast array of GPCRs both in vitro and in vivo.

Allosteric Regulation↗
PubMed2004

Proliferation of microglia, but not photoreceptors, in the outer nuclear layer of the rd-1 mouse.

PURPOSE: To establish whether photoreceptor apoptosis in the rd-1 mouse is accompanied by cell cycle progression. Studies of cell cycle proteins in other models of neuronal death provide consistent evidence that a repertoire of proliferative markers accompanies apoptosis. METHODS: The spatiotemporal progression of photoreceptor loss in rd-1 and control mice at postnatal days (PN)8, -10, -12, -15, and -18 was correlated with markers of G(1)- and S-phase progression. Photoreceptor death was detected by using morphology and terminal dUTP transferase nick end labeling (TUNEL). Cell-cycle-associated markers consisted of bromodeoxyuridine (BrdU) uptake, and immunolabeling for proliferating cell nuclear antigen (PCNA), Ki-67, and cyclin-dependent kinases-2 and -4. The identity of proliferating cells in the outer nuclear layer was established by double immunolabeling with PCNA and either F4/80 or recoverin. RESULTS: A population of proliferating cells in the outer nuclear layer accompanies photoreceptor death along a central to peripheral gradient in rd-1 retinas. Double immunolabeling for PCNA and F4/80 readily identified these as microglial cells originating from the inner retina. Cell cycle progression in photoreceptors could not be demonstrated. CONCLUSIONS: These findings confirm that in rd-1, a preexisting condition for cell cycle progression does not exist as it does in other neurodegenerative conditions. Therefore, in this model, evidence of photoreceptor cell cycle progression in retinas exposed to neurotrophic factors is likely to result from the therapy itself. In addition, the results confirmed that proliferating microglial cells are intimately associated with the degenerative process in rd-1.

Animals↗
PubMed2004

Otx2 homeobox gene induces photoreceptor-specific phenotypes in cells derived from adult iris and ciliary tissue.

PURPOSE: It remains unclear which gene induction effectively generates photoreceptor-specific phenotypes from nonretinal tissues. The purpose of this study was to determine whether Crx and Otx2--homeobox genes related to photoreceptor development--can induce the generation of these phenotypes in cells derived from adult ciliary and iris tissue and in mesencephalon-derived neural stem cells. METHODS: Crx and Otx2 were transferred into adult rat ciliary- and embryonic mesencephalon-derived neurospheres and adult rat iris-derived cells with the aid of a recombinant retrovirus. The presence of photoreceptor-specific phenotypes was confirmed by immunocytochemistry and Western blot analysis. RESULTS: More than 90% of the Crx- and Otx2-transfected ciliary- and iris-derived cells exhibited rod opsin immunoreactivity, whereas few of the similarly transfected mesencephalon-derived neural stem cells expressed rod opsin. At least two additional key components of the phototransduction cascade, recoverin and Gdeltat1, were expressed by Crx- and Otx2-transfected iris-derived cells. CONCLUSIONS: Crx and Otx2 effectively induced the generation of photoreceptor-specific phenotypes from ciliary- and iris-derived cells. That both Crx and Otx2 induced phenotype generation in cells derived from iris or ciliary tissue may suggest an approach to photoreceptor cell preparation for retinal transplantation.

Animals↗