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Sökning: WFRF:(Paquet Durand François)

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1.
  • Arango-Gonzalez, Blanca, et al. (författare)
  • Identification of a common non-apoptotic cell death mechanism in hereditary retinal degeneration.
  • 2014
  • Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:11
  • Tidskriftsartikel (refereegranskat)abstract
    • Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments.
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2.
  • Azadi, Seifollah, et al. (författare)
  • CNTF plus BDNF treatment and neuroprotective pathways in the rd1 mouse retina
  • 2007
  • Ingår i: Brain Research. - : Elsevier BV. - 1872-6240 .- 0006-8993. ; 1129:1, s. 116-129
  • Tidskriftsartikel (refereegranskat)abstract
    • The rd1 mouse is a relevant model for studying the mechanisms of photoreceptor degeneration in retinitis pigmentosa. Treatment with ciliary neurotrophic factor (CNTF) in combination with brain derived neurotrophic factor (BDNF) is known to rescue photoreceptors in cultured rd1 retinal explants. To shed light on the underlying mechanisms, we studied the effects of 9 days (starting at postnatal day 2) in vitro CNTF+BDNF treatment on the endogenous production of CNTF, BDNF, fibroblast growth factor 2 (FGF2), or the activation of extracellular signal-regulated kinase (ERK), Akt and CAMP-response-element-binding protein (CREB) in retinal explants. In rd1 explants, CNTF+BDNF decreased the number of TUNEL-positive photoreceptors. The treatment also increased endogenous rd1 levels of CNTF and BDNF, but lowered the level of FGF2 expression in rd1 explants. When wild-type explants were treated, endogenous CNTF was similarly increased, while BDNF and FGF2 levels remained unaffected. In addition, treatment of rd1 retinas strongly increased the phosphorylation of ERK, Akt and CREB. In treated wild-type explants, the same parameters were either unchanged (ERK) or decreased (Akt and CREB). The results suggest a role for Akt, ERK and CREB in conveying the neuroprotective effect of CNTF+BDNF treatment in rd1 retinal explants. (c) 2006 Elsevier B.V. All rights reserved.
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3.
  • Azadi, Seifollah, et al. (författare)
  • Up-regulation and increased phosphorylation of protein kinase C (PKC) delta, mu and theta in the degenerating rd1 mouse retina.
  • 2006
  • Ingår i: Molecular and Cellular Neuroscience. - : Elsevier BV. - 1044-7431. ; 31:4, s. 759-773
  • Tidskriftsartikel (refereegranskat)abstract
    • The rd1 mouse serves as a model for inherited photoreceptor degeneration: retinitis pigmentosa. Microarray techniques were employed to compare the transcriptomes of rd1 and congenic wild-type retinas at postnatal day 11, when degenerative processes have started but most photoreceptors are still present. Of the several genes that were differentially expressed, focus was put on those associated with the protein kinase C (PKC) signaling pathway, in particular PKCδ, μ and θ. Microarray identified these as being up-regulated in the rd1 retina, which was confirmed by QRT-PCR. Western blotting and immunostaining, using antibodies against either total or phosphorylated variants of the PKC isoforms, revealed increased expression and phosphorylation of PKCδ, μ and θ in the rd1 retina at the protein level as well. Our results suggest that these PKC isoforms are involved in rd1 degeneration.
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4.
  • Bicker, Gerd, et al. (författare)
  • Simulation of stroke-related damage in cultured human nerve cells
  • 2007
  • Ingår i: ALTEX Alternatives To Animal Experimentation. - 0946-7785. ; 24, s. 16-18
  • Tidskriftsartikel (refereegranskat)abstract
    • We describe a novel cell culture protocol for the generation of neurons from a human teratocarcinoma cell line. These neurons were used to investigate hypoxic-ischaemic cell damage and for developing neuroprotective strategies. Cultures of human model neurons should eventually serve to reduce the number of experimental animals in cerebral stroke research.
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5.
  • Bujakowska, Kinga, et al. (författare)
  • Study of Gene-Targeted Mouse Models of Splicing Factor Gene Prpf31 Implicated in Human Autosomal Dominant Retinitis Pigmentosa (RP)
  • 2009
  • Ingår i: Investigative Ophthalmology & Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 1552-5783. ; 50:12, s. 5927-5933
  • Tidskriftsartikel (refereegranskat)abstract
    • PURPOSE. Pre-mRNA processing factor 31 (PRPF31) is a ubiquitous protein needed for the assembly of the pre-mRNA splicing machinery. It has been shown that mutations in this gene cause autosomal dominant retinitis pigmentosa 11 (RP11), which is characterized by rod-cell degeneration. Interestingly, mutations in this ubiquitously expressed gene do not lead to phenotypes other than retinal malfunction. Furthermore, the dominant inheritance pattern has shown incomplete penetrance, which poses interesting questions about the disease mechanism of RP11. METHODS. To characterize PRPF31 function in the rod cells, two animal models have been generated. One was a heterozygous knock-in mouse (Prpf31(A216P/+)) carrying a point mutation p.A216P, which has previously been identified in RP11 patients. The second was a heterozygous knockout mouse (Prpf31(+/-)). Retinal degeneration in RP11 mouse models was monitored by electroretinography and histology. RESULTS. Generation of the mouse models is presented, as are results of ERGs and retinal morphology. No degenerative phenotype on fundus examination was found in Prpf31(A216P/+) and Prpf31(+/-) mice. Prpf31(A216P/A216P) and Prpf31(-/-) genotypes were embryonic lethal. CONCLUSIONS. The results imply that Prpf31 is necessary for survival, and there is no compensation mechanism in mouse for the lack of this splicing factor. The authors suggest that p.A216P mutation in Prpf31 does not exert a dominant negative effect and that one Prpf31 wild-type allele is sufficient for maintenance of the healthy retina in mice.
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6.
  • Christensen, Gustav, et al. (författare)
  • Investigating Ex Vivo Animal Models to Test the Performance of Intravitreal Liposomal Drug Delivery Systems
  • 2021
  • Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 13:7
  • Tidskriftsartikel (refereegranskat)abstract
    • There is a strong need for innovative and efficient drug delivery systems for ocular therapy development. However, testing intravitreal drug delivery systems without using live animals is challenging. Ex vivo animal models offer an interesting alternative. We analyzed the potential of using fresh porcine eyes obtained from the local slaughterhouse as a model for testing the intravitreal biodistribution and retention of liposomes with or without polyethylene glycol (PEG) conjugation and with different surface charges. The histology of the eyes was analyzed to localize the liposomes, and it was found that liposomes with PEG absorbed rapidly on the retina (within 1 h), with positively charged and PEG-coated liposomes being retained for at least 24 h. In parallel, fluorophotometry was employed on intact eyes, to determine the pharmacokinetics of the fluorophore calcein, as a substitute for a small hydrophilic therapeutic compound. We found a 4.5-fold increase in the vitreous half-life of calcein loaded in liposomes, compared with the free solution. Retinal toxicity was addressed using murine-derived retinal explant cultures. Liposomes were non-toxic up to 500 µg/mL. Toxicity was observed at 5 mg/mL for anionic and cationic liposomes, with 2-fold and 2.5-fold increased photoreceptor cell death, respectively. Overall, we could show that important ocular drug delivery considerations such as pharmacokinetics and biodistribution can be estimated in ex vivo porcine eyes, and may guide subsequent in vivo experiments.
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7.
  • Christensen, Gustav, et al. (författare)
  • Ocular permeability, intraocular biodistribution of lipid nanocapsule formulation intended for retinal drug delivery
  • 2023
  • Ingår i: European journal of pharmaceutics and biopharmaceutics. - : Elsevier B.V.. - 0939-6411 .- 1873-3441. ; 187, s. 175-183
  • Tidskriftsartikel (refereegranskat)abstract
    • Recently, cGMP analogues have been investigated for the treatment of inherited retinal degenerations (IRD) using intravitreal injections. However, higher vitreous elimination rates limit the possibility to treat the retina with small molecule drugs. Here, we investigated the potential of lipid nanocapsules (LNCs) as vehicles to reduce clearance and prolong the delivery of cGMP analogue, CN03 to the retinal photoreceptors. Initially LNCs were investigated for both topical/periocular and intravitreal administration routes. While LNC-mediated drug permeation through the cornea proved to be too low for clinical applications, intravitreal application showed significant promise. Intravitreally administered LNCs containing fluorescent tracer in ex vivo porcine eyes showed complete intravitreal dispersal within 24 h. Ocular bio-distribution on histological sections showed that around 10 % of the LNCs had reached the retina, and 40 % accumulated in the ciliary body. For comparison, we used fluorescently labeled liposomes and these showed a different intraocular distribution with 48 % accumulated in the retina, and almost none were in the ciliary body. LNCs were then tested in retinal explants prepared from wild-type (WT) and rd1 mouse. In WT retina LNCs showed no significant toxic effects up to a concentration of 5 mg/mL. In rd1 retina, the LNC/CN03 formulation protected rd1 photoreceptors with similar efficacy to that of free CN03, demonstrating the usefulness of LNC/CN03 formulation in the treatment of IRD. Overall, our results indicate the suitability of LNCs for intraocular administration and drug delivery to both the retina and the ciliary body. © 2023 The Author(s)
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8.
  • Christensen, Gustav, et al. (författare)
  • Pyruvate-conjugation of PEGylated liposomes for targeted drug delivery to retinal photoreceptors
  • 2023
  • Ingår i: Biomedicine and Pharmacotherapy. - : Elsevier Masson s.r.l.. - 0753-3322 .- 1950-6007. ; 163
  • Tidskriftsartikel (refereegranskat)abstract
    • Despite several promising candidates, there is a paucity of drug treatments available for patients suffering from retinal diseases. An important reason for this is the lack of suitable delivery systems that can achieve sufficiently high drug uptake in the retina and its photoreceptors. A promising and versatile method for drug delivery to specific cell types involves transporter-targeted liposomes, i.e., liposomes surface-coated with substrates for transporter proteins highly expressed on the target cell. We identified strong lactate transporter (monocarboxylate transporter, MCT) expression on photoreceptors as a potential target for drug delivery vehicles. To evaluate MCT suitability for drug targeting, we used PEG-coated liposomes and conjugated these with different monocarboxylates, including lactate, pyruvate, and cysteine. Monocarboxylate-conjugated and dye-loaded liposomes were tested on both human-derived cell-lines and murine retinal explant cultures. We found that liposomes conjugated with pyruvate consistently displayed higher cell uptake than unconjugated liposomes or liposomes conjugated with lactate or cysteine. Pharmacological inhibition of MCT1 and MCT2 reduced internalization, suggesting an MCT-dependent uptake mechanism. Notably, pyruvate-conjugated liposomes loaded with the drug candidate CN04 reduced photoreceptor cell death in the murine rd1 retinal degeneration model while free drug solutions could not achieve the same therapeutic effect. Our study thus highlights pyruvate-conjugated liposomes as a promising system for drug delivery to retinal photoreceptors, as well as other neuronal cell types displaying high expression of MCT-type proteins. © 2023 The Authors
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9.
  • Farinelli, Pietro, et al. (författare)
  • DNA methylation and differential gene regulation in photoreceptor cell death.
  • 2014
  • Ingår i: Cell Death & Disease. - : Springer Science and Business Media LLC. - 2041-4889. ; 5
  • Tidskriftsartikel (refereegranskat)abstract
    • Retinitis pigmentosa (RP) defines a group of inherited degenerative retinal diseases causing progressive loss of photoreceptors. To this day, RP is still untreatable and rational treatment development will require a thorough understanding of the underlying cell death mechanisms. Methylation of the DNA base cytosine by DNA methyltransferases (DNMTs) is an important epigenetic factor regulating gene expression, cell differentiation, cell death, and survival. Previous studies suggested an involvement of epigenetic mechanisms in RP, and in this study, increased cytosine methylation was detected in dying photoreceptors in the rd1, rd2, P23H, and S334ter rodent models for RP. Ultrastructural analysis of photoreceptor nuclear morphology in the rd1 mouse model for RP revealed a severely altered chromatin structure during retinal degeneration that coincided with an increased expression of the DNMT isozyme DNMT3a. To identify disease-specific differentially methylated DNA regions (DMRs) on a genomic level, we immunoprecipitated methylated DNA fragments and subsequently analyzed them with a targeted microarray. Genome-wide comparison of DMRs between rd1 and wild-type retina revealed hypermethylation of genes involved in cell death and survival as well as cell morphology and nervous system development. When correlating DMRs with gene expression data, we found that hypermethylation occurred alongside transcriptional repression. Consistently, motif analysis showed that binding sites of several important transcription factors for retinal physiology were hypermethylated in the mutant model, which also correlated with transcriptional silencing of their respective target genes. Finally, inhibition of DNMTs in rd1 organotypic retinal explants using decitabine resulted in a substantial reduction of photoreceptor cell death, suggesting inhibition of DNA methylation as a potential novel treatment in RP.
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10.
  • Farinelli, Pietro, et al. (författare)
  • Retinitis Pigmentosa: Over-expression of anti-ageing protein Klotho in degenerating photoreceptors.
  • 2013
  • Ingår i: Journal of Neurochemistry. - : Wiley. - 1471-4159 .- 0022-3042.
  • Tidskriftsartikel (refereegranskat)abstract
    • Retinitis Pigmentosa involves a hereditary degeneration of photoreceptors by as yet unresolved mechanisms. The secretable protein α-Klotho has a function related to ageing processes, and α-Klotho deficient mice have reduced lifespan and declining functions in several tissues. Here, we studied Klotho in connection with inherited photoreceptor degeneration. Increased nuclear immunostaining for α-Klotho protein was seen in degenerating photoreceptors in four different Retinitis Pigmentosa models (rd1, rd2 mice; P23H, S334ter rhodopsin mutant rats). Correspondingly, in rd1 retina α-Klotho mRNA expression was significantly upregulated. Moreover, immunostaining for another Klotho family protein, β-Klotho, also co-localised with degenerating rd1 photoreceptors. The rd1 retina displayed reduced levels of fibroblast growth factor (FGF) 15, a member of the FGF subfamily for which Klotho acts as a co-receptor. Exogenous α-Klotho protein added to retinal explant cultures did not affect cell death in rd1 retinae, but caused a severe layer disordering in wild-type retinae. Our work suggests Klotho as a novel player in the retina, with a clear connection to photoreceptor cell death as well as with an influence on retinal organization. This article is protected by copyright. All rights reserved.
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11.
  • Hauck, Stefanie M, et al. (författare)
  • Differential modification of phosducin protein in degenerating rd1 ret is associated with constitutively active CaMKII in rod outer segments.
  • 2006
  • Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 5:2, s. 324-336
  • Tidskriftsartikel (refereegranskat)abstract
    • Retinitis pigmentosa comprises a heterogeneous group of incurable progressive blinding diseases with unknown pathogenic mechanisms. The retinal degeneration 1 (rd1) mouse is a retinitis pigmentosa model that carries a mutation in a rod photoreceptor-specific phosphodiesterase gene, leading to rapid degeneration of these cells. Elucidation of the molecular differences between rd1 and healthy retinae is crucial for explaining this degeneration and could assist in suggesting novel therapies. Here we used high resolution proteomics to compare the proteomes of the rd1 mouse retina and its congenic, wildtype counterpart at postnatal day 11 when photoreceptor death is profound. Over 3000 protein spots were consistently resolved by two-dimensional gel electrophoresis and subjected to a rigorous filtering procedure involving computer-based spot analyses. Five proteins were accepted as being differentially expressed in the rd1 model and subsequently identified by mass spectrometry. The difference in one such protein, phosducin, related to an altered modification pattern in the rd1 retina rather than to changed expression levels. Additional experiments showed phosducin in healthy retinae to be highly phosphorylated in the dark- but not in the light-adapted phase. In contrast, rd1 phosducin was highly phosphorylated irrespective of light status, indicating a dysfunctional rd1 light/dark response. The increased rd1 phosducin phosphorylation coincided with increased activation of calcium/calmodulin-activated protein kinase II, which is known to utilize phosducin as a substrate. Given the increased rod calcium levels present in the rd1 mutation, calcium-evoked overactivation of this kinase may be an early and long sought for step in events leading to photoreceptor degeneration in the rd1 mouse.
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12.
  • Himawan, Erico, et al. (författare)
  • Drug delivery to retinal photoreceptors
  • 2019
  • Ingår i: Drug Discovery Today. - : Elsevier BV. - 1359-6446. ; 24:8, s. 1637-1643
  • Forskningsöversikt (refereegranskat)abstract
    • The photoreceptors of the retina are afflicted by diseases that still often lack satisfactory treatment options. Although suitable drugs might be available in some cases, the delivery of these compounds into the eye and across the blood–retinal barrier remains a significant challenge for therapy development. Here, we review the routes of drug administration to the retina and highlight different options for drug delivery to the photoreceptor cells.
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13.
  • Huang, Li, et al. (författare)
  • Efficient Delivery of Hydrophilic Small Molecules to Retinal Cell Lines Using Gel Core-Containing Solid Lipid Nanoparticles
  • 2022
  • Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 14:1, s. 74-74
  • Tidskriftsartikel (refereegranskat)abstract
    • In this study, we developed a novel solid lipid nanoparticle (SLN) formulation for drug delivery of small hydrophilic cargos to the retina. The new formulation, based on a gel core and composite shell, allowed up to two-fold increase in the encapsulation efficiency. The type of hydrophobic polyester used in the composite shell mixture affected the particle surface charge, colloidal stability, and cell internalization profile. We validated SLNs as a drug delivery system by performing the encapsulation of a hydrophilic neuroprotective cyclic guanosine monophosphate analog, previously demonstrated to hold retinoprotective properties, and the best formulation resulted in particles with a size of ±250 nm, anionic charge > −20 mV, and an encapsulation efficiency of ±60%, criteria that are suitable for retinal delivery. In vitro studies using the ARPE-19 and 661W retinal cell lines revealed the relatively low toxicity of SLNs, even when a high particle concentration was used. More importantly, SLN could be taken up by the cells and the release of the hydrophilic cargo in the cytoplasm was visually demonstrated. These findings suggest that the newly developed SLN with a gel core and composite polymer/lipid shell holds all the characteristics suitable for the drug delivery of small hydrophilic active molecules into retinal cells.
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14.
  • Kaur, Jasvir, et al. (författare)
  • Calpain and PARP Activation during Photoreceptor Cell Death in P23H and S334ter Rhodopsin Mutant Rats
  • 2011
  • Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:7
  • Tidskriftsartikel (refereegranskat)abstract
    • Retinitis pigmentosa (RP) is a heterogeneous group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness. Many human cases are caused by mutations in the rhodopsin gene. An important question regarding RP pathology is whether different genetic defects trigger the same or different cell death mechanisms. To answer this question, we analysed photoreceptor degeneration in P23H and S334ter transgenic rats carrying rhodopsin mutations that affect protein folding and sorting respectively. We found strong activation of calpain and poly(ADP-ribose) polymerase (PARP) in both mutants, concomitant with calpastatin down-regulation, increased oxidative DNA damage and accumulation of PAR polymers. These parameters were strictly correlated with the temporal progression of photoreceptor degeneration, mirroring earlier findings in the phosphodiesterase-6 mutant rd1 mouse, and suggesting execution of non-apoptotic cell death mechanisms. Interestingly, activation of caspases-3 and -9 and cytochrome c leakage-key events in apoptotic cell death-were observed only in the S334ter mutant, which also showed increased expression of PARP-1. The identification of the same metabolic markers triggered by different mutations in two different species suggests the existence of common cell death mechanisms, which is a major consideration for any mutation independent treatment.
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15.
  • Kuehlewein, Laura, et al. (författare)
  • Clinical phenotype and course of PDE6A-associated retinitis pigmentosa disease, characterized in preparation for a gene supplementation trial
  • 2020
  • Ingår i: JAMA Ophthalmology. - : American Medical Association (AMA). - 2168-6165. ; 138:12, s. 1241-1250
  • Tidskriftsartikel (refereegranskat)abstract
    • IMPORTANCE Treatment trials require sound knowledge on the natural course of disease. OBJECTIVE To assess clinical features, genetic findings, and genotype-phenotype correlations in patients with retinitis pigmentosa (RP) associated with biallelic sequence variations in the PDE6A gene in preparation for a gene supplementation trial. DESIGN, SETTING, AND PARTICIPANTS This prospective, longitudinal, observational cohort study was conducted from January 2001 to December 2019 in a single center (Centre for Ophthalmology of the University of Tübingen, Germany) with patients recruited multinationally from 12 collaborating European tertiary referral centers. Patients with retinitis pigmentosa, sequence variants in PDE6A, and the ability to provide informed consent were included. EXPOSURES Comprehensive ophthalmological examinations; validation of compound heterozygosity and biallelism by familial segregation analysis, allelic cloning, or assessment of next-generation sequencing-read data, where possible. MAIN OUTCOMES AND MEASURES Genetic findings and clinical features describing the entire cohort and comparing patients harboring the 2 most common disease-causing variants in a homozygous state (c.304C>A;p.(R102S) and c.998 + 1G>A;p.?). RESULTS Fifty-seven patients (32 female patients [56%]; mean [SD], 40 [14] years) from 44 families were included. All patients completed the study. Thirty patients were homozygous for disease-causing alleles. Twenty-seven patients were heterozygous for 2 different PDE6A variants each. The most frequently observed alleles were c.304C>A;p.(R102S), c.998 + 1G>A;p.?, and c.2053G>A;p.(V685M). The mean (SD) best-corrected visual acuity was 0.43 (0.48) logMAR (Snellen equivalent, 20/50). The median visual field area with object III4e was 660 square degrees (5th and 95th percentiles, 76 and 11 019 square degrees; 25th and 75th percentiles, 255 and 3923 square degrees). Dark-adapted and light-adapted full-field electroretinography showed no responses in 88 of 108 eyes (81.5%). Sixty-nine of 108 eyes (62.9%) showed additional findings on optical coherence tomography imaging (eg, cystoid macular edema or macular atrophy). The variant c.998 + 1G>A;p.? led to a more severe phenotype when compared with the variant c.304C>A;p.(R102S). CONCLUSIONS AND RELEVANCE Seventeen of the PDE6A variants found in these patients appeared to be novel. Regarding the clinical findings, disease was highly symmetrical between the right and left eyes and visual impairment was mild or moderate in 90% of patients, providing a window of opportunity for gene therapy.
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16.
  • Marigo, Valeria, et al. (författare)
  • Modulation of cGMP-signalling to Prevent Retinal Degeneration
  • 2019
  • Ingår i: RSC Drug Discovery Series. - Cambridge : Royal Society of Chemistry. - 2041-3203. ; 2019-January:66, s. 88-98
  • Tidskriftsartikel (refereegranskat)abstract
    • In the photoreceptors of the retina, the second-messenger molecule cyclic guanosine monophosphate (cGMP) occupies centre stage in the phototransduction cascade. Remarkably, cGMP is also involved in hereditary photoreceptor degeneration caused by a variety of different genetic insults. This provides an entry point for the development of inhibitory cGMP analogues for a mutation-independent treatment. Here, we outline how cGMP signalling can be targeted for the treatment of retinal degeneration, how inhibitory cGMP analogues may be designed and formulated, and how test systems of rising complexity can be used to identify new compounds with photoreceptor neuroprotective properties. In this context, we cite the European Union-funded DRUGSFORD project and provide an example for the efficacy of a specific cGMP analogue to prevent photoreceptor loss and preserve retinal function.
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17.
  • Paquet-Durand, Francois, et al. (författare)
  • A key role for cyclic nucleotide gated (CNG) channels in cGMP-related retinitis pigmentosa
  • 2011
  • Ingår i: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 20:5, s. 941-947
  • Tidskriftsartikel (refereegranskat)abstract
    • The rd1 natural mutant is one of the first and probably the most commonly studied mouse model for retinitis pigmentosa (RP), a severe and frequently blinding human retinal degeneration. In several decades of research, the link between the increase in photoreceptor cGMP levels and the extremely rapid cell death gave rise to a number of hypotheses. Here, we provide clear evidence that the presence of cyclic nucleotide gated (CNG) channels in the outer segment membrane is the key to rod photoreceptor loss. In Cngb1(-/-) x rd1 double mutants devoid of regular CNG channels, cGMP levels are still pathologically high, but rod photoreceptor viability and outer segment morphology are greatly improved. Importantly, cone photoreceptors, the basis for high-resolution daylight and colour vision, survived and remained functional for extended periods of time. These findings strongly support the hypothesis of deleterious calcium (Ca2+)-influx as the cause of rapid rod cell death and highlight the importance of CNG channels in this process. Furthermore, our findings suggest that targeting rod CNG channels, rather than general Ca2+-channel blockade, is a most promising symptomatic approach to treat otherwise incurable forms of cGMP-related RP.
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18.
  • Paquet-Durand, Francois, et al. (författare)
  • Calpain activity in retinal degeneration.
  • 2007
  • Ingår i: Journal of Neuroscience Research. - : Wiley. - 1097-4547 .- 0360-4012. ; 85:4, s. 693-702
  • Forskningsöversikt (refereegranskat)abstract
    • Retinal degenerations such as retinitis pigmentosa (RP) or glaucoma are a major cause of blindness in humans. Understanding the mechanisms underlying the various types of retinal degeneration is a pre-requisite for the development of rational therapies for these diseases. Activation of the calcium dependent protease, calpain, has been suggested to play an important role in cell death in various neuronal tissues including the retina. Improved detection and analysis of calpain activity during degenerative processes is likely to expand the list of pathological conditions with calpain involvement. We give a short overview of the methods available for the detection of calpain activity, and briefly discuss properties of calpain inhibitors. We then discuss the role of calpains in different cell death mechanisms and review existing work on retinal degeneration and the possible involvement of calpains therein. The implication of calpains in retinal cell death raises the possibility to use calpain inhibitors to prevent or delay retinal degeneration. (c) 2006 Wiley-Liss, Inc.
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19.
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20.
  • Paquet-Durand, Francois, et al. (författare)
  • Diltiazem protects human NT-2 neurons against excitotoxic damage in a model of simulated ischemia
  • 2006
  • Ingår i: Brain Research. - : Elsevier BV. - 1872-6240 .- 0006-8993. ; 1124, s. 45-54
  • Tidskriftsartikel (refereegranskat)abstract
    • in vitro models are often used to investigate pathophysiological mechanisms of brain cell injury as they occur for instance during cerebral ischemia. To analyze the efficacy of potential neuroprotective compounds, cell physiological experiments were performed in a recently improved culture system of human model neurons. The postmitotic neurons were generated from the human NT-2 teratocarcinoma cell line, using a cell sphere culture method to facilitate rapid terminal differentiation. We simulated ischemic conditions in cultures of purified NT-2 neurons and found that low doses of the antihypertensive drug diltiazem protected against excitotoxic neuronal damage in vitro. Experiments with primary cortical mouse neuron cultures demonstrated a similar response to simulated ischemia and confirmed the neuroprotective effect of diltiazem. Calcium imaging experiments showed that diltiazem reduced both NMDA- and glutamate-induced calcium influxes in NT-2 neurons suggesting that its neuroprotective effect is based on the inhibition of voltage-gated calcium channels. These results indicate that diltiazem is an effective blocker of glutamate-induced excitotoxicity. Moreover, we suggest that cell cultures of human model neurons can provide an important initial test system for drug development in stroke therapy. (c) 2006 Elsevier B.V. All rights reserved.
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21.
  • Paquet-Durand, Francois, et al. (författare)
  • Human model neurons in studies of brain cell damage and neural repair.
  • 2007
  • Ingår i: Current Molecular Medicine. - 1566-5240. ; 7:6, s. 541-554
  • Forskningsöversikt (refereegranskat)abstract
    • Disorders of the central nervous system are a major concern in modern human societies. Studies of these disorders require the use of suitable model systems that accurately reproduce the human situation. In this article we focus on the possibilities of using the human NT-2 teratocarcinoma cell line for studies on neuronal differentiation, cellular function and neurodegeneration. Neurons generated from undifferentiated NT-2 precursor cells show neuronal morphology, express neuronal markers, exhibit action potentials and have the advantage of homogeneous cellular composition of clonally derived cells. They release a number of different neurotransmitters, respond to stimulation with glutamate, gamma-amino-butyric acid, and nitric oxide, and form functional synapses in culture. Depending on the differentiation protocol, NT-2 cells also have the capacity to develop into glial cells. Different neuronal differentiation procedures and biological properties of NT-2 neurons are described in the text. In transplantation experiments, differentiated NT-2 neurons integrated successfully into the nervous systems of both experimental animals and human patients without evidence for tumor formation, underlining their value for both basic research and clinical applications. We discuss some potential applications in the fields of basic research, drug discovery, and therapy of CNS damage with particular emphasis on neuronal transplantation and different cell death mechanisms in neuronal degeneration. Grafting of NT-2 neurons has been shown to effectively reverse functional defects in animal disease models. Moreover, an ongoing phase 2 randomized clinical trial indicates the safety and feasibility of NT2 neuron transplantation for the treatment of human patients with cerebral stroke.
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22.
  • Paquet-Durand, François, et al. (författare)
  • Modulation of Calcium Overload and Calpain Activity
  • 2019
  • Ingår i: RSC Drug Discovery Series. - Cambridge : Royal Society of Chemistry. - 2041-3203. ; 2019-January:66, s. 48-60
  • Tidskriftsartikel (refereegranskat)abstract
    • Fluxes of calcium ions (Ca2+) in rod photoreceptors are major regulators of steady-state and light-evoked intracellular reactions to stimuli. The homeostasis of Ca2+ is regulated by channels and pumps localized at the plasma membrane and in intracellular organelles. Photoreceptor degeneration is frequently associated with Ca2+ homeostasis disruption and stimulation of Ca2+ activated proteases, such as calpains. These events trigger molecular pathways leading to cell death. In this chapter we discuss Ca2+ channels and pumps as well as calpains as potential targets of new therapies for retinal degeneration.
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23.
  • Paquet-Durand, Francois, et al. (författare)
  • Photoreceptor rescue and toxicity induced by different calpain inhibitors
  • 2010
  • Ingår i: Journal of Neurochemistry. - : Wiley. - 1471-4159 .- 0022-3042. ; 115:4, s. 930-940
  • Tidskriftsartikel (refereegranskat)abstract
    • P>Photoreceptor degeneration is the hallmark of a group of inherited blinding diseases collectively termed retinitis pigmentosa (RP); a major cause of blindness in humans. RP is at present untreatable and the underlying neurodegenerative mechanisms are largely unknown, even though the genetic causes are often established. The activation of calpain-type proteases may play an important role in cell death in various neuronal tissues, including the retina. We therefore tested the efficacy of two different calpain inhibitors in preventing cell death in the retinal degeneration (rd1) human homologous mouse model for RP. Pharmacological inhibition of calpain activity in rd1 organotypic retinal explants had ambiguous effects on photoreceptor viability. Calpain inhibitor XI had protective effects when applied for short periods of time (16 h) but demonstrated substantial levels of toxicity in both wild-type and rd1 retina when used over several days. In contrast, the highly specific calpain inhibitor calpastatin peptide reduced photoreceptor cell death in vitro after both short and prolonged exposure, an effect that was also evident after in vivo application via intravitreal injection. These findings highlight the importance of calpain activation for photoreceptor cell death but also for photoreceptor survival and propose the use of highly specific calpain inhibitors to prevent or delay RP.
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24.
  • Paquet-Durand, Francois, et al. (författare)
  • PKG activity causes photoreceptor cell death in two retinitis pigmentosa models
  • 2009
  • Ingår i: Journal of Neurochemistry. - : Wiley. - 1471-4159 .- 0022-3042. ; 108:3, s. 796-810
  • Tidskriftsartikel (refereegranskat)abstract
    • Photoreceptor degeneration in retinitis pigmentosa is one of the leading causes of hereditary blindness in the developed world. Although causative genetic mutations have been elucidated in many cases, the underlying neuronal degeneration mechanisms are still unknown. Here, we show that activation of cGMP-dependent protein kinase (PKG) hallmarks photoreceptor degeneration in rd1 and rd2 human homologous mouse models. When induced in wild-type retinae, PKG activity was both necessary and sufficient to trigger cGMP-mediated photoreceptor cell death. Target-specific, pharmacological inhibition of PKG activity in both rd1 and rd2 retinae strongly reduced photoreceptor cell death in organotypic retinal explants. Likewise, inhibition of PKG in vivo, using three different application paradigms, resulted in robust photoreceptor protection in the rd1 retina. These findings suggest a pivotal role for PKG activity in cGMP-mediated photoreceptor degeneration mechanisms and highlight the importance of PKG as a novel target for the pharmacological intervention in RP.
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25.
  • Paquet-Durand, Francois, et al. (författare)
  • Turning teratocarcinoma cells into neurons: rapid differentiation of NT-2 cells in floating spheres
  • 2003
  • Ingår i: Developmental Brain Research. - 0165-3806. ; 142:2, s. 161-167
  • Tidskriftsartikel (refereegranskat)abstract
    • Cells from the human teratocarcinoma line NTera-2 can be induced to terminally differentiate into postmitotic neurons when treated with retinoic acid. However, this differentiation process is rather time consuming as it takes between 42 and 54 days. Here, we propose a modified differentiation protocol which reduces the time needed for differentiation considerably without compromising the quantity of the neurons obtained. The introduction of a proliferation step as free floating cell spheres cuts the total time needed to obtain high yields of purified NT-2 neurons to about 24-28 days. The cells obtained show neuronal morphology and migrate to form ganglion-like cell conglomerates. Differentiated cells express neuronal polarity markers such as the cytoskeleton associated proteins MAP2 and Tau. Moreover, the generation of neurons in sphere cultures induced immunoreactivity to the ELAV-like neuronal RNA-binding proteins HuC/D, which have been implicated in mechanisms of nerve cell differentiation.
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26.
  • Power, Michael, et al. (författare)
  • Cellular mechanisms of hereditary photoreceptor degeneration – Focus on cGMP
  • 2020
  • Ingår i: Progress in Retinal and Eye Research. - : Elsevier BV. - 1350-9462. ; 74
  • Forskningsöversikt (refereegranskat)abstract
    • The cellular mechanisms underlying hereditary photoreceptor degeneration are still poorly understood, a problem that is exacerbated by the enormous genetic heterogeneity of this disease group. However, the last decade has yielded a wealth of new knowledge on degenerative pathways and their diversity. Notably, a central role of cGMP-signalling has surfaced for photoreceptor cell death triggered by a subset of disease-causing mutations. In this review, we examine key aspects relevant for photoreceptor degeneration of hereditary origin. The topics covered include energy metabolism, epigenetics, protein quality control, as well as cGMP- and Ca2+-signalling, and how the related molecular and metabolic processes may trigger photoreceptor demise. We compare and integrate evidence on different cell death mechanisms that have been associated with photoreceptor degeneration, including apoptosis, necrosis, necroptosis, and PARthanatos. A special focus is then put on the mechanisms of cGMP-dependent cell death and how exceedingly high photoreceptor cGMP levels may cause activation of Ca2+-dependent calpain-type proteases, histone deacetylases and poly-ADP-ribose polymerase. An evaluation of the available literature reveals that a large group of patients suffering from hereditary photoreceptor degeneration carry mutations that are likely to trigger cGMP-dependent cell death, making this pathway a prime target for future therapy development. Finally, an outlook is given into technological and methodological developments that will with time likely contribute to a comprehensive overview over the entire metabolic complexity of photoreceptor cell death. Building on such developments, new imaging technology and novel biomarkers may be used to develop clinical test strategies, that fully consider the genetic heterogeneity of hereditary retinal degenerations, in order to facilitate clinical testing of novel treatment approaches.
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27.
  • Rasmussen, Michel, et al. (författare)
  • The photoreceptor protective cGMP-analog Rp-8-Br-PET-cGMPS interacts with cGMP-interactors PKGI, PDE1, PDE6, and PKAI in the degenerating mouse retina
  • 2023
  • Ingår i: Journal of Comparative Neurology. - : Wiley. - 0021-9967 .- 1096-9861. ; 531:8, s. 935-951
  • Tidskriftsartikel (refereegranskat)abstract
    • The inherited eye disease retinitis pigmentosa (RP) causes the loss of photoreceptors by a still unknown cell death mechanism. During this degeneration, cyclic guanosine-3′,5′-monophosphate (cGMP) levels become elevated, leading to over-activation of the cGMP-binding protein cGMP-dependent protein kinase (PKG). cGMP analogs selectively modified to have inhibitory actions on PKG have aided in impeding photoreceptor death, and one such cGMP analog is Rp-8-Br-PET-cGMPS. However, cGMP analogs have previously been shown to interact with numerous targets, so to better understand the therapeutic action of Rp-8-Br-PET-cGMPS, it is necessary to elucidate its target-selectivity and hence what potential cellular mechanism(s) it may affect within the photoreceptors. Here, we, therefore, applied affinity chromatography together with mass spectrometry to isolate and identify Rp-8-Br-PET-cGMPS interactors from retinas derived from three different murine RP models (i.e., rd1, rd2, and rd10 mice). Our findings revealed that Rp-8-Br-PET-cGMPS bound seven known cGMP-binding proteins, including PKG1β, PDE1β, PDE1c, PDE6α, and PKA1α. Furthermore, an additional 28 proteins were found to be associated with Rp-8-Br-PET-cGMPS. This latter group included MAPK1/3, which is known to connect with cGMP/PKG in other systems. However, in organotypic retinal cultures, Rp-8-Br-PET-cGMPS had no effect on photoreceptor MAPK1/3 expression or activity. To summarize, Rp-8-Br-PET-cGMPS is more target specific compared to regular cGMP.
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28.
  • Roy, Akanksha, et al. (författare)
  • Integrative Kinase Activity Profiling and Phosphoproteomics of rd10 Mouse Retina during cGMP-Dependent Retinal Degeneration
  • 2024
  • Ingår i: International Journal of Molecular Sciences. - 1661-6596. ; 25:6
  • Tidskriftsartikel (refereegranskat)abstract
    • Inherited retinal degenerative diseases (IRDs) are a group of rare diseases that lead to a progressive loss of photoreceptor cells and, ultimately, blindness. The overactivation of cGMP-dependent protein kinase G (PKG), one of the key effectors of cGMP-signaling, was previously found to be involved in photoreceptor cell death and was studied in murine IRD models to elucidate the pathophysiology of retinal degeneration. However, PKG is a serine/threonine kinase (STK) with several hundred potential phosphorylation targets and, so far, little is known about the specificity of the target interaction and downstream effects of PKG activation. Here, we carried out both the kinome activity and phosphoproteomic profiling of organotypic retinal explant cultures derived from the rd10 mouse model for IRD. After treating the explants with the PKG inhibitor CN03, an overall decrease in peptide phosphorylation was observed, with the most significant decrease occurring in seven peptides, including those from the known PKG substrate cyclic-AMP-response-element-binding CREB, but also Ca2+/calmodulin-dependent kinase (CaMK) peptides and TOP2A. The phosphoproteomic data, in turn, revealed proteins with decreased phosphorylation, as well as proteins with increased phosphorylation. The integration of both datasets identified common biological networks altered by PKG inhibition, which included kinases predominantly from the so-called AGC and CaMK families of kinases (e.g., PKG1, PKG2, PKA, CaMKs, RSKs, and AKTs). A pathway analysis confirmed the role of CREB, Calmodulin, mitogen-activated protein kinase (MAPK) and CREB modulation. Among the peptides and pathways that showed reduced phosphorylation activity, the substrates CREB, CaMK2, and CaMK4 were validated for their retinal localization and activity, using immunostaining and immunoblotting in the rd10 retina. In summary, the integrative analysis of the kinome activity and phosphoproteomic data revealed both known and novel PKG substrates in a murine IRD model. This data establishes a basis for an improved understanding of the biological pathways involved in cGMP-mediated photoreceptor degeneration. Moreover, validated PKG targets like CREB and CaMKs merit exploration as novel (surrogate) biomarkers to determine the effects of a clinical PKG-targeted treatment for IRDs.
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29.
  • Sahaboglu, A, et al. (författare)
  • Knockout of PARG110 confers resistance to cGMP-induced toxicity in mammalian photoreceptors.
  • 2014
  • Ingår i: Cell Death & Disease. - : Springer Science and Business Media LLC. - 2041-4889. ; 5:May 22
  • Tidskriftsartikel (refereegranskat)abstract
    • Hereditary retinal degeneration (RD) relates to a heterogeneous group of blinding human diseases in which the light sensitive neurons of the retina, the photoreceptors, die. RD is currently untreatable and the underlying cellular mechanisms remain poorly understood. However, the activity of the enzyme poly-ADP-ribose polymerase-1 (PARP1) and excessive generation of poly-ADP-ribose (PAR) polymers in photoreceptor nuclei have been shown to be causally involved in RD. The activity of PARP1 is to a large extent governed by its functional antagonist, poly-ADP-glycohydrolase (PARG), which thus also may have a role in RD. To investigate this, we analyzed PARG expression in the retina of wild-type (wt) mice and in the rd1 mouse model for human RD, and detected increased PARG protein in a subset of degenerating rd1 photoreceptors. Knockout (KO) animals lacking the 110 kDa nuclear PARG isoform were furthermore analyzed, and their retinal morphology and function were indistinguishable from wild-type animals. Organotypic wt retinal explants can be experimentally treated to induce rd1-like photoreceptor death, but PARG110 KO retinal explants were unexpectedly highly resistant to such treatment. The resistance was associated with decreased PAR accumulation and low PARP activity, indicating that PARG110 may positively regulate PARP1, an event that therefore is absent in PARG110 KO tissue. Our study demonstrates a causal involvement of PARG110 in the process of photoreceptor degeneration. Contrasting its anticipated role as a functional antagonist, absence of PARG110 correlated with low PARP activity, suggesting that PARG110 and PARP1 act in a positive feedback loop, which is especially active under pathologic conditions. This in turn highlights both PARG110 and PARP1 as potential targets for neuroprotective treatments for RD.
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30.
  • Sahaboglu, Ayse, et al. (författare)
  • PARP1 Gene Knock-Out Increases Resistance to Retinal Degeneration without Affecting Retinal Function
  • 2010
  • Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:11
  • Tidskriftsartikel (refereegranskat)abstract
    • Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness in humans. Previously, excessive activation of enzymes belonging to the poly-ADP-ribose polymerase (PARP) group was shown to be involved in photoreceptor degeneration in the human homologous rd1 mouse model for RP. Since there are at least 16 different PARP isoforms, we investigated the exact relevance of the predominant isoform - PARP1 - for photoreceptor cell death using PARP1 knock-out (KO) mice. In vivo and ex vivo morphological analysis using optic coherence tomography (OCT) and conventional histology revealed no major alterations of retinal phenotype when compared to wild-type (wt). Likewise, retinal function as assessed by electroretinography (ERG) was normal in PARP1 KO animals. We then used retinal explant cultures derived from wt, rd1, and PARP1 KO animals to test their susceptibility to chemically induced photoreceptor degeneration. Since photoreceptor degeneration in the rd1 retina is triggered by a loss-of-function in phosphodiesterase-6 (PDE6), we used selective PDE6 inhibition to emulate the rd1 situation on non-rd1 genotypes. While wt retina subjected to PDE6 inhibition showed massive photoreceptor degeneration comparable to rd1 retina, in the PARP1 KO situation, cell death was robustly reduced. Together, these findings demonstrate that PARP1 activity is in principle dispensable for normal retinal function, but is of major importance for photoreceptor degeneration under pathological conditions. Moreover, our results suggest that PARP dependent cell death or PARthanatos may play a major role in retinal degeneration and highlight the possibility to use specific PARP inhibitors for the treatment of RP.
  •  
31.
  • Vighi, Eleonora, et al. (författare)
  • Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration
  • 2018
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 115:13, s. 2997-3006
  • Tidskriftsartikel (refereegranskat)abstract
    • Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of “druggable” targets, and the access-limiting blood–retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3′,5′-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.
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