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1.
J Fish Biol ; 101(5): 1361-1365, 2022 Nov.
Article in English | MEDLINE | ID: mdl-35906859

ABSTRACT

The authors investigated left-right turning preferences of n = 260 juvenile European sea bass (Dicentrarchus labrax) reared in ambient conditions and ocean acidification (OA) conditions or in ambient conditions but tested in OA water. Groups of 10 individuals were observed alone in a circular tank, and individuals' left and right turning during free-swimming was quantified using trajectory data from the video. The authors showed that near-future OA levels do not affect the number of turns made, or behavioural lateralization (turning preference), in juvenile D. labrax tested in groups.


Subject(s)
Bass , Animals , Swimming , Carbon Dioxide , Hydrogen-Ion Concentration , Seawater
2.
J Vis Exp ; (126)2017 08 12.
Article in English | MEDLINE | ID: mdl-28829422

ABSTRACT

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.


Subject(s)
Cell Transplantation/methods , Retinal Pigment Epithelium/transplantation , Transplantation, Heterologous/methods , Animals , Humans , Injections, Intraocular/methods , Macular Degeneration/therapy , Rats , Retina/transplantation , Retinal Pigment Epithelium/cytology
3.
Stem Cell Reports ; 9(1): 42-49, 2017 07 11.
Article in English | MEDLINE | ID: mdl-28625537

ABSTRACT

Age-related macular degeneration (AMD) is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE) cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC)-derived RPE cells (RPESC-RPE) preserved vision in a rat model of RPE cell dysfunction. Importantly, the stage of differentiation that RPESC-RPE acquired prior to transplantation influenced the efficacy of vision rescue. Whereas cells at all stages of differentiation tested rescued photoreceptor layer morphology, an intermediate stage of RPESC-RPE differentiation obtained after 4 weeks of culture was more consistent at vision rescue than progeny that were differentiated for 2 weeks or 8 weeks of culture. Our results indicate that the developmental stage of RPESC-RPE significantly influences the efficacy of RPE cell replacement, which affects the therapeutic application of these cells for AMD.


Subject(s)
Adult Stem Cells/cytology , Cell Differentiation , Macular Degeneration/therapy , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/transplantation , Animals , Cell Culture Techniques , Cells, Cultured , Humans , Macular Degeneration/pathology , Rats , Retinal Pigment Epithelium/pathology , Swine , Vision, Ocular
4.
J Ocul Pharmacol Ther ; 32(5): 304-9, 2016 06.
Article in English | MEDLINE | ID: mdl-27182605

ABSTRACT

PURPOSE: Numerous preclinical studies have shown that transplantation of stem cell-derived retinal pigment epithelial cell (RPE) preserves photoreceptor cell anatomy in the dystrophic Royal College of Surgeons (RCS) rat. How rescue is spatially distributed over the eye, relative to the transplantation site, is less clear. To understand spatial variations in transplant efficacy, we have developed a method to measure the spatial distribution of rescued photoreceptor cells. METHODS: Human RPE Stem Cell-derived RPE (RPESC-RPE) cells were subretinally injected into RCS rat eyes. After tissue recovery and orientating the globe, a series of retinal sections were cut through the injected area. Sections were stained with DAPI (4',6-diamidino-2-phenylindole) and a number of photoreceptor nuclei were counted across the nasal-temporal and superior-inferior axes. These data were used to construct 2D maps of the area of photoreceptor cell saving. RESULTS: Photoreceptor cell preservation was detected in the injected temporal hemisphere and occupied areas greater than 4 mm(2) centered near the injection sites. Rescue was directed toward the central retina and superior and inferior poles, with maximal number of rescued photoreceptor cells proximal to the injection sites. CONCLUSIONS: RPESC-RPE transplantation preserves RCS photoreceptor cells. The photoreceptor cell contour maps readily convey the extent of rescue across the eye. The consistent alignment and quantification of results using this method allow the application of other downstream statistical analyses and comparisons to better understand transplantation therapy in the eye.


Subject(s)
Photoreceptor Cells, Vertebrate , Retinal Pigment Epithelium/cytology , Stem Cells , Animals , Humans , Rats , Rats, Long-Evans , Rats, Mutant Strains
5.
Cancer Res ; 75(10): 1992-2004, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25769723

ABSTRACT

Prostate cancer is the second leading form of cancer-related death in men. In a subset of prostate cancer patients, increased chemokine signaling IL8 and IL6 correlates with castrate-resistant prostate cancer (CRPC). IL8 and IL6 are produced by prostate epithelial cells and promote prostate cancer cell invasion; however, the mechanisms restraining prostate epithelial cell cytokine secretion are poorly understood. Herein, the cell-fate determinant factor DACH1 inhibited CRPC tumor growth in mice. Using Dach1(fl/fl)/Probasin-Cre bitransgenic mice, we show IL8 and IL6 secretion was altered by approximately 1,000-fold by endogenous Dach1. Endogenous Dach1 is shown to serve as a key endogenous restraint to prostate epithelial cell growth and restrains migration via CXCL signaling. DACH1 inhibited expression, transcription, and secretion of the CXCL genes (IL8 and IL6) by binding to their promoter regulatory regions in chromatin. DACH1 is thus a newly defined determinant of benign and malignant prostate epithelium cellular growth, migration, and cytokine abundance in vivo.


Subject(s)
Cell Movement , Cytokines/metabolism , Epithelial Cells/physiology , Eye Proteins/physiology , Prostatic Neoplasms/metabolism , Transcription Factors/physiology , Animals , Apoptosis , Cell Line, Tumor , Cell Proliferation , Humans , Male , Mice, Transgenic , Neoplasm Transplantation , Prostate/pathology , Prostatic Neoplasms/pathology , Protein Sorting Signals
6.
Bioorg Med Chem Lett ; 24(9): 2212-21, 2014 May 01.
Article in English | MEDLINE | ID: mdl-24703233

ABSTRACT

In this Letter, we present the results of a hit-finding and lead optimization programme against the EP4 receptor (EP4R). In a short time period, we were able to discover five structurally diverse series of hit compounds using a combination of virtual screening methods. The most favoured hit, compound 6, was demonstrated to be a competitive antagonist of the EP4R. Compound 73 was identified following several rounds of optimization, which centred on improving both the primary EP4R affinity and selectivity against the related EP2R as well as the aqueous solubility. This work culminated in the preparation of PGN-1531, the sodium salt of 73, which showed a marked improvement in solubility (>10 mg/mL). PGN-1531 is a potent and selective antagonist at EP4Rs in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation.


Subject(s)
Drug Discovery , Receptors, Prostaglandin E, EP4 Subtype/antagonists & inhibitors , Brain/blood supply , HEK293 Cells , Humans , Ligands , Migraine Disorders/drug therapy , Models, Molecular , Receptors, Prostaglandin E, EP4 Subtype/metabolism , Vasodilation/drug effects
7.
Graefes Arch Clin Exp Ophthalmol ; 252(3): 423-31, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24492934

ABSTRACT

PURPOSE: The objective of this study was to determine whether cells from the conjunctiva could be reprogrammed into induced pluripotent stem (iPS) cells, providing an alternative source of stem cells. METHODS: We employed a doxycycline-induced reprogrammable mouse strain to generate iPS cells from conjunctiva. The identity of the stem cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assays. Immunocytochemistry and teratoma assays are established means for scoring stem cell pluripotency. The reprogramming efficiencies of conjunctival cells and ear fibroblasts were compared. RESULTS: We confirmed the identity of the stem cells and demonstrated expression of pluripotency markers (OCT4, SOX2, NANOG, and SSEA1), as tested by RT-PCR and immunofluorescence assays. In addition, derived iPS cells differentiated successfully into embryoid bodies, and showed teratoma formation when injected into immunodeficient mice. Reprogramming conjunctival tissue is as efficient as reprogramming ear fibroblasts. Conjunctiva-iPS exhibited classic features of embryonic stem (ES) cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, capacity to differentiate in vitro, and the ability to form all three germ layers in vivo. CONCLUSION: The present study demonstrated that conjunctival cells, which are readily obtained during the course of many routine conjunctival biopsies and ophthalmic procedures, can be another reliable source of iPS cells.


Subject(s)
Conjunctiva/cytology , Induced Pluripotent Stem Cells/cytology , Animals , Anti-Bacterial Agents/pharmacology , Biomarkers/metabolism , Cell Culture Techniques , Coculture Techniques , Conjunctiva/drug effects , Conjunctiva/metabolism , Doxycycline/pharmacology , Fibroblasts/cytology , Fluorescent Antibody Technique, Indirect , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Induced Pluripotent Stem Cells/metabolism , Karyotyping , Lewis X Antigen/genetics , Lewis X Antigen/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nanog Homeobox Protein , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism
8.
Trans Am Ophthalmol Soc ; 112: 103-15, 2014 Jul.
Article in English | MEDLINE | ID: mdl-25646031

ABSTRACT

PURPOSE: To assess the functional consequences of silencing of tuberin, an inhibitor of the mTOR signaling pathway, in a preclinical model of retinitis pigmentosa (RP) in order to test the hypothesis that insufficient induction of the protein kinase B (PKB)-regulated tuberin/mTOR self-survival pathway initiates apoptosis. METHODS: In an unbiased genome-scale approach, kinase peptide substrate arrays were used to analyze self-survival pathways at the onset of photoreceptor degeneration. The mutant Pde6b(H620Q)/Pde6b(H620Q) at P14 and P18 photoreceptor outer segment (OS) lysates were labeled with P-ATP and hybridized to an array of 1,164 different synthetic peptide substrates. At this stage, OS of Pde6b(H620Q)/Pde6b(H620Q) rods are morphologically normal. In vitro kinase assays and immunohistochemistry were used to validate phosphorylation. Short hairpin RNA (shRNA) gene silencing was used to validate tuberin's role in regulating survival. RESULTS: At the onset of degeneration, 162 peptides were differentially phosphorylated. Protein kinases A, G, C (AGC kinases), and B exhibited increased activity in both peptide array and in vitro kinase assays. Immunohistochemical data confirmed altered phosphorylation patterns for phosphoinositide-dependent kinase-1 (PDK1), ribosomal protein S6 (RPS6), and tuberin. Tuberin gene silencing rescued photoreceptors from degeneration. CONCLUSIONS: Phosphorylation of tuberin and RPS6 is due to the upregulated activity of PKB. PKB/tuberin cell growth/survival signaling is activated before the onset of degeneration. Substrates of the AGC kinases in the PKB/tuberin pathway are phosphorylated to promote cell survival. Knockdown of tuberin, the inhibitor of the mTOR pathway, increased photoreceptor survival and function in a preclinical model of RP.


Subject(s)
Retinitis Pigmentosa/physiopathology , Tumor Suppressor Proteins/physiology , Animals , Apoptosis/physiology , Disease Models, Animal , Gene Silencing , Mice , Mice, Mutant Strains , Phosphorylation , Phosphotransferases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Ribosomal Protein S6/physiology , Signal Transduction/physiology , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/genetics
9.
J Neurosci ; 33(33): 13475-83, 2013 Aug 14.
Article in English | MEDLINE | ID: mdl-23946405

ABSTRACT

The third-most common cause of autosomal recessive retinitis pigmentosa (RP) is due to defective cGMP phosphodiesterase-6 (PDE6). Previous work using viral gene therapy on PDE6-mutant mouse models demonstrated photoreceptors can be rescued if administered before degeneration. However, whether visual function can be rescued after degeneration onset has not been addressed. This is a clinically important question, as newly diagnosed patients exhibit considerable loss of rods and cones in their peripheral retinas. We have generated and characterized a tamoxifen inducible Cre-loxP rescue allele, Pde6b(Stop), which allows us to temporally correct PDE6-deficiency. Whereas untreated mutants exhibit degeneration, activation of Cre-loxP recombination in early embryogenesis produced stable long-term rescue. Reversal at later time-points showed partial long-term or short-lived rescue. Our results suggest stable restoration of retinal function by gene therapy can be achieved if a sufficient number of rods are treated. Because patients are generally diagnosed after extensive loss of rods, the success of clinical trials may depend on identifying patients as early as possible to maximize the number of treatable rods.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 6/therapeutic use , Genetic Therapy/methods , Retinitis Pigmentosa/genetics , Animals , Cyclic Nucleotide Phosphodiesterases, Type 6/genetics , Disease Models, Animal , Electroretinography , Mice , Mice, Inbred C57BL , Mice, Mutant Strains
10.
Hum Mol Genet ; 22(3): 558-67, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23108158

ABSTRACT

Approximately 36 000 cases of simplex and familial retinitis pigmentosa (RP) worldwide are caused by a loss in phosphodiesterase (PDE6) function. In the preclinical Pde6α(nmf363) mouse model of this disease, defects in the α-subunit of PDE6 result in a progressive loss of photoreceptors and neuronal function. We hypothesized that increasing PDE6α levels using an AAV2/8 gene therapy vector could improve photoreceptor survival and retinal function. We utilized a vector with the cell-type-specific rhodopsin (RHO) promoter: AAV2/8(Y733F)-Rho-Pde6α, to transduce Pde6α(nmf363) retinas and monitored its effects over a 6-month period (a quarter of the mouse lifespan). We found that a single injection enhanced survival of photoreceptors and improved retinal function. At 6 months of age, the treated eyes retained photoreceptor cell bodies, while there were no detectable photoreceptors remaining in the untreated eyes. More importantly, the treated eyes demonstrated functional visual responses even after the untreated eyes had lost all vision. Despite focal rescue of the retinal structure adjacent to the injection site, global functional rescue of the entire retina was observed. These results suggest that RP due to PDE6α deficiency in humans, in addition to PDE6ß deficiency, is also likely to be treatable by gene therapy.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 6/genetics , Genetic Therapy/methods , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/therapy , Animals , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Dependovirus/genetics , Disease Models, Animal , Electroretinography , Genetic Vectors , Immunoblotting , Mice , Mice, Inbred C57BL , Mice, Transgenic , Promoter Regions, Genetic , Retina/anatomy & histology , Retina/physiopathology , Retinitis Pigmentosa/physiopathology , Rhodopsin/genetics , Rhodopsin/metabolism , Transduction, Genetic
11.
J Vis Exp ; (69)2012 Nov 25.
Article in English | MEDLINE | ID: mdl-23207897

ABSTRACT

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.(1) Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.(2) These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.(3-4) Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.(5-8) In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.


Subject(s)
Genetic Therapy/methods , Genetic Vectors/administration & dosage , Retina/physiology , Animals , Embryonic Stem Cells/physiology , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Lentivirus/genetics , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Stem Cell Transplantation
12.
PLoS One ; 7(8): e43889, 2012.
Article in English | MEDLINE | ID: mdl-22970106

ABSTRACT

Vigabatrin (VGB) is a commonly prescribed antiepileptic drug designed to inhibit GABA-transaminase, effectively halting seizures. Unfortunately, VGB treatment is also associated with the highest frequencies of peripheral visual field constriction of any of the antiepileptic drugs and the mechanisms that lead to these visual field defects are uncertain. Recent studies have demonstrated light exposure exacerbates vigabatrin-induced retinal toxicity. We further assessed this relationship by examining the effects of vigabatrin treatment on the retinal structures of mice with genetically altered photoreception. In keeping with previous studies, we detected increased toxicity in mice exposed to continuous light. To study whether cone or rod photoreceptor function was involved in the pathway to toxicity, we tested mice with mutations in the cone-specific Gnat2 or rod-specific Pde6g genes, and found the mutations significantly reduced VGB toxicity. Our results confirm light is a significant enhancer of vigabatrin toxicity and that a portion of this is mediated, directly or indirectly, by phototransduction signaling in rod and cone photoreceptors.


Subject(s)
Anticonvulsants/toxicity , Retinal Cone Photoreceptor Cells/drug effects , Rod Cell Outer Segment/drug effects , Vigabatrin/toxicity , Vision, Ocular/drug effects , Animals , Dose-Response Relationship, Drug , Light , Mice , Mice, Transgenic , Retina/abnormalities , Retina/drug effects , Retina/physiopathology , Retinal Cone Photoreceptor Cells/physiology , Retinal Cone Photoreceptor Cells/radiation effects , Rod Cell Outer Segment/physiology , Rod Cell Outer Segment/radiation effects
14.
Mol Med ; 18: 1312-9, 2012 Dec 06.
Article in English | MEDLINE | ID: mdl-22895806

ABSTRACT

The U.S. Food and Drug Administration recently approved phase I/II clinical trials for embryonic stem (ES) cell-based retinal pigmented epithelium (RPE) transplantation, but this allograft transplantation requires lifelong immunosuppressive therapy. Autografts from patient-specific induced pluripotent stem (iPS) cells offer an alternative solution to this problem. However, more data are required to establish the safety and efficacy of iPS transplantation in animal models before moving iPS therapy into clinical trials. This study examines the efficacy of iPS transplantation in restoring functional vision in Rpe65(rd12)/Rpe65(rd12) mice, a clinically relevant model of retinitis pigmentosa (RP). Human iPS cells were differentiated into morphologically and functionally RPE-like tissue. Quantitative real-time polymerase chain reaction (RT-PCR) and immunoblots confirmed RPE fate. The iPS-derived RPE cells were injected into the subretinal space of Rpe65(rd12)/Rpe65(rd12) mice at 2 d postnatally. After transplantation, the long-term surviving iPS-derived RPE graft colocalized with the host native RPE cells and assimilated into the host retina without disruption. None of the mice receiving transplants developed tumors over their lifetimes. Furthermore, electroretinogram, a standard method for measuring efficacy in human trials, demonstrated improved visual function in recipients over the lifetime of this RP mouse model. Our study provides the first direct evidence of functional recovery in a clinically relevant model of retinal degeneration using iPS transplantation and supports the feasibility of autologous iPS cell transplantation for retinal and macular degenerations featuring significant RPE loss.


Subject(s)
Induced Pluripotent Stem Cells/cytology , Retinitis Pigmentosa/therapy , Stem Cell Transplantation/adverse effects , Animals , Biomarkers/metabolism , Cell Differentiation , Cell Line , Cell Survival , Cell Transformation, Neoplastic/pathology , Cellular Reprogramming , Disease Models, Animal , Electroretinography , Fibroblasts/cytology , Humans , Mice , Mice, SCID , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/physiopathology , Retinal Pigment Epithelium/transplantation , Retinal Pigment Epithelium/ultrastructure , Retinitis Pigmentosa/physiopathology , Skin/cytology , Time Factors , Treatment Outcome
15.
Cell Signal ; 24(1): 181-8, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21920434

ABSTRACT

The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and ß subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g(tm1)/Pde6g(tm1) mice that do not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile(86) and Ile(87) residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and ß by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100µM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile(86) and Ile(87) residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 6/genetics , Retinal Rod Photoreceptor Cells/metabolism , Sequence Deletion , Signal Transduction , 1-Methyl-3-isobutylxanthine/pharmacology , Algorithms , Animals , Cyclic Nucleotide Phosphodiesterases, Type 6/antagonists & inhibitors , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Eye Proteins/antagonists & inhibitors , Eye Proteins/metabolism , Female , Kinetics , Light , Male , Membrane Potentials/radiation effects , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Retina/metabolism , Retina/pathology , Retinal Degeneration/genetics , Retinal Degeneration/pathology , Retinal Rod Photoreceptor Cells/drug effects
16.
Exp Biol Med (Maywood) ; 236(10): 1211-7, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21885480

ABSTRACT

Mutations in Pde6b lead to high levels of signaling molecules cyclic guanosine monophosphate (cGMP) and Ca(2+), which ultimately result in photoreceptor cell death in certain forms of retinitis pigmentosa (RP). The level of cGMP, which is controlled by opposing activities of guanylate cyclase (GUCY) and photoreceptor phosphodiesterase-6 (PDE6), regulates the opening of cyclic nucleotide-gated ion channels [CNG] and thereby controls Ca(2+) influx into the outer segments. Using a lentiviral gene therapy approach, we have previously shown that degeneration can be temporarily slowed either by introducing wild-type PDE6ß or knocking down expression of GUCY2E and CNGA1 in photoreceptors of Pde6b(H620Q), a mouse model for RP. Rescue was transient with either approach. Therefore, we tested a novel combination therapy using bipartite lentiviral vectors designed to both introduce wild-type PDE6ß expression and knockdown GUCY2E or CNGA1. Immunoblot analysis shows simultaneous increases in PDE6ß and decreases in GUCY2E or CNGA1 in retinas transduced by the vectors, indicating successful transduction. In Pde6b(H620Q) mutants, we observe rescue of photoreceptor function and an increase in photoreceptor rows as compared with untreated controls. However, no evidence of prolonged rescue beyond the limit of the previously tested single therapy was observed.


Subject(s)
DNA, Complementary/therapeutic use , Genetic Therapy/methods , Lentivirus/genetics , RNA, Small Interfering/therapeutic use , Retinitis Pigmentosa/therapy , Animals , Cyclic Nucleotide Phosphodiesterases, Type 6/genetics , Genetic Vectors/genetics , Genetic Vectors/therapeutic use , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Photoreceptor Cells, Vertebrate/physiology , Retina/metabolism , Retina/virology , Retinitis Pigmentosa/genetics , Transduction, Genetic/methods
17.
J Cell Mol Med ; 15(8): 1778-87, 2011 Aug.
Article in English | MEDLINE | ID: mdl-20950332

ABSTRACT

In vertebrate rods, dark and light conditions produce changes in guanosine 3',5'-cyclic monophosphate (cGMP) and calcium (Ca(2+) ) levels, which are regulated by the opposing function of several proteins. During the recovery of a bright flash, guanylate cyclase (GUCY) helps raise cGMP to levels that open cGMP-gated calcium sodium channels (CNG) to increase Na(+) and Ca(2+) influx in the outer segment. In contrast, light activates cGMP phosphodiesterase 6 (PDE6) causing rapid hydrolysis of cGMP, CNG closure, and reduced Na(+) and Ca(2+) levels. In Pde6b mouse models of retinitis pigmentosa (RP), photoreceptor death is preceded by abnormally high cGMP and Ca(2+) levels, likely because of continued synthesis of cGMP by guanylate cyclases and unregulated influx of Ca(2+) to toxic levels through CNG channels. To reverse the effects of Pde6b loss of function, we employed an shRNA knockdown approach to reduce the expression of Gucy2e or Cnga1 in Pde6b(H620Q) photoreceptors prior to degeneration. Gucy2e- or Cnga1-shRNA lentiviral-mediated knockdown GUCY2E and CNGA1 expression increase visual function and photoreceptor survival in Pde6b(H620Q) mice. We demonstrated that effective knockdown of GUCY2E and CNGA1 expression to counteract loss of PDE6 function may develop into a valuable approach for treating some patients with RP.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Cyclic Nucleotide-Gated Cation Channels/metabolism , Guanylate Cyclase/metabolism , Photoreceptor Cells, Vertebrate/metabolism , Receptors, Cell Surface/metabolism , Retinitis Pigmentosa/metabolism , Animals , Base Sequence , Calcium/metabolism , Cell Survival , Cyclic GMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 6/genetics , Cyclic Nucleotide-Gated Cation Channels/genetics , Disease Models, Animal , Electroretinography , Female , Guanylate Cyclase/genetics , Humans , Immunoblotting , Light , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Photoreceptor Cells, Vertebrate/pathology , RNA Interference , Receptors, Cell Surface/genetics , Retina/metabolism , Retina/physiopathology , Retina/radiation effects , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/physiopathology , Sodium/metabolism
18.
Biochem Biophys Res Commun ; 390(4): 1149-53, 2009 Dec 25.
Article in English | MEDLINE | ID: mdl-19878658

ABSTRACT

The gamma subunit of rod-specific cGMP phosphodiesterase 6 (PDE6gamma), an effector of the G-protein GNAT1, is a key regulator of phototransduction. The results of several in vitro biochemical reconstitution experiments conducted to examine the effects of phosphorylation of PDE6gamma on its ability to regulate the PDE6 catalytic core have been inconsistent, showing that phosphorylation of PDE6gamma may increase or decrease the ability of PDE6gamma to deactivate phototransduction. To resolve role of phosphorylation of PDE6gamma in living photoreceptors, we generated transgenic mice in which either one or both Threonine (T) sites in PDE6gamma (T22 and T35), which are candidates for putative regulatory phosphorylation, were substituted with alanine (A). Phosphorylation of these sites was examined as a function of light exposure. We found that phosphorylation of T22 increases with light exposure in intact mouse rods while constitutive phosphorylation of T35 is unaffected by light in intact mouse rods and cones. Phosphorylation of the cone isoform of PDE6gamma, PDE6H, is constitutively phosphorylated at the T20 residue. Light-induced T22 phosphorylation was lost in T35A transgenic rods, and T35 phosphorylation was extinguished in T22A transgenic rods. The interdependency of phosphorylation of T22 and T35 suggests that light-induced, post-translational modification of PDE6gamma is essential for the regulation of G-protein signaling.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Light , Protein Processing, Post-Translational , Retinal Cone Photoreceptor Cells/radiation effects , Retinal Rod Photoreceptor Cells/radiation effects , Animals , Antibodies, Phospho-Specific/immunology , Cattle , GTP-Binding Protein alpha Subunits/metabolism , Light Signal Transduction , Mice , Mice, Inbred Strains , Mice, Transgenic , Phosphorylation , Retinal Cone Photoreceptor Cells/enzymology , Retinal Rod Photoreceptor Cells/enzymology , Signal Transduction , Threonine/immunology , Threonine/metabolism , Transducin/metabolism
19.
Eur Neuropsychopharmacol ; 19(3): 196-204, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19116182

ABSTRACT

Blockade of the cannabinoid CB1 receptors (CB1R) has been shown to reduce psychostimulant-induced hyperactivity, an effect that we sought to further characterize here. The CB1R antagonist SR141716A dose-dependently decreased d-amphetamine-induced hyperactivity.Also, d-amphetamine-induced hyperlocomotion was reduced in CB1R knockout (KO) mice. However, CB1R KO and wild-type mice showed a similar d-amphetamine-induced increase in nucleus accumbens DA release. Hence, we investigated whether CB1R antagonism/invalidation reduces d-amphetamine-induced hyperlocomotion through a mechanism involving changes in glutamatergic neurotransmission. Blockade of metabotropic-glutamate-receptors-5 (mGluR5)with MPEP, but not blockade of N-methyl-D-aspartate-receptors (NMDA) with MK-801,restored to a great extent the blunted d-amphetamine-induced hyperlocomotion seen after CB1R antagonism/invalidation. Thus, hyporesponsiveness to the psychostimulant effects of d-amphetamine as a result of CB1R antagonism/invalidation is not due to an ensuing decrease in d-amphetamine-induced DA release in the nucleus accumbens, but rather due to a hyperglutamatergic state and facilitation of glutamatergic neurotransmission at the mGlu5, but not NMDA, receptors.


Subject(s)
Amphetamine/pharmacology , Behavior, Animal/drug effects , Dopamine Uptake Inhibitors/pharmacology , Receptor, Cannabinoid, CB1/deficiency , Receptors, Metabotropic Glutamate/metabolism , Analysis of Variance , Animals , Dizocilpine Maleate/pharmacology , Dopamine/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Hyperkinesis/chemically induced , Hyperkinesis/drug therapy , Hyperkinesis/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microdialysis/methods , Motor Activity/drug effects , Nucleus Accumbens/drug effects , Piperidines/pharmacology , Pyrazoles/pharmacology , Pyridines/pharmacology , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Metabotropic Glutamate 5 , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Rimonabant
20.
Synapse ; 62(12): 940-3, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18798268

ABSTRACT

We investigated the participation of the metabotropic glutamate receptor type 5 (mGluR5) in mediating increases in cortical acetylcholine (ACh) efflux elicited by established or putative neuropsychotherapeutic compounds, using in vivo microdialysis in rats. The norepinephrine transporter inhibitor atomoxetine, the cannabinoid CB1 receptor antagonist SR141716A, the dopamine D1 receptor agonist dihydrexidine, and the atypical antipsychotic clozapine increased cortical ACh (by about 2-3 fold), whereas the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) by itself had no effect. The stimulatory effects of atomoxetine, SR141716A and dihydrexidine on cortical ACh were abolished by pretreatment with MPEP. MPEP also attenuated the stimulatory effect of clozapine on ACh efflux. Thus, mGluR5 activation appears to be involved in the procholinergic effects of compounds that exhibit therapeutic properties or potential in neuropsychiatry.


Subject(s)
Acetylcholine/physiology , Cholinergic Agents/pharmacology , Psychotropic Drugs/pharmacology , Receptors, Metabotropic Glutamate/physiology , Animals , Cerebral Cortex/drug effects , Cerebral Cortex/physiology , Rats , Receptor, Metabotropic Glutamate 5
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