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1.
Glia ; 71(11): 2511-2526, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37533369

RESUMO

The expressions of ion channels by Müller glial cells (MGCs) may change in response to various retinal pathophysiological conditions. There remains a gap in our understanding of MGCs' responses to photoreceptor degeneration towards finding therapies. The study explores how an inhibition of store-operated Ca2+ entry (SOCE) and its major component, Orai1 channel, in MGCs protects photoreceptors from degeneration. The study revealed increased Orai1 expression in the MGCs of retinal degeneration 10 (rd10) mice. Enhanced expression of oxidative stress markers was confirmed as a crucial pathological mechanism in rd10 retina. Inducing oxidative stress in rat MGCs resulted in increasing SOCE and Ca2+ release-activated Ca2+ (CRAC) currents. SOCE inhibition by 2-Aminoethoxydiphenyl borate (2-APB) protected photoreceptors in degenerated retinas. Finally, molecular simulations proved the structural and dynamical features of 2-APB to the target structure Orai1. Our results provide new insights into the physiology of MGCs regarding retinal degeneration and shed a light on SOCE and Orai1 as new therapeutic targets.


Assuntos
Canais de Cálcio , Degeneração Retiniana , Ratos , Camundongos , Animais , Canais de Cálcio/metabolismo , Células Ependimogliais/metabolismo , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/prevenção & controle , Cálcio/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Sinalização do Cálcio/fisiologia
2.
Cell Mol Life Sci ; 77(11): 2199-2216, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31451894

RESUMO

The enzyme poly-ADP-ribose-polymerase (PARP) has important roles for many forms of DNA repair and it also participates in transcription, chromatin remodeling and cell death signaling. Currently, some PARP inhibitors are approved for cancer therapy, by means of canceling DNA repair processes and cell division. Drug repurposing is a new and attractive aspect of therapy development that could offer low-cost and accelerated establishment of new treatment options. Excessive PARP activity is also involved in neurodegenerative diseases including the currently untreatable and blinding retinitis pigmentosa group of inherited retinal photoreceptor degenerations. Hence, repurposing of known PARP inhibitors for patients with non-oncological diseases might provide a facilitated route for a novel retinitis pigmentosa therapy. Here, we demonstrate and compare the efficacy of two different PARP inhibitors, BMN-673 and 3-aminobenzamide, by using a well-established retinitis pigmentosa model, the rd1 mouse. Moreover, the mechanistic aspects of the PARP inhibitor-induced protection were also investigated in the present study. Our results showed that rd1 rod photoreceptor cell death was decreased by about 25-40% together with the application of these two PARP inhibitors. The wealth of human clinical data available for BMN-673 highlights a strong potential for a rapid clinical translation into novel retinitis pigmentosa treatments. Remarkably, we have found that the efficacy of 3 aminobenzamide was able to decrease PARylation at the nanomolar level. Our data also provide a link between PARP activity with the Wnt/ß-catenin pathway and the major intracellular antioxidant concentrations behind the PARP-dependent retinal degeneration. In addition, molecular modeling studies were integrated with experimental studies for better understanding of the role of PARP1 inhibitors in retinal degeneration.


Assuntos
Benzamidas/uso terapêutico , Ftalazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Degeneração Retiniana/tratamento farmacológico , Retinose Pigmentar/tratamento farmacológico , Animais , Reposicionamento de Medicamentos/métodos , Humanos , Camundongos , Poli(ADP-Ribose) Polimerases/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia
3.
Proc Natl Acad Sci U S A ; 115(13): E2997-E3006, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29531030

RESUMO

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.


Assuntos
Barreira Hematorretiniana/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/administração & dosagem , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Degeneração Retiniana/tratamento farmacológico , Animais , Barreira Hematorretiniana/efeitos dos fármacos , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Lipossomos , Camundongos , Células Fotorreceptoras/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Degeneração Retiniana/metabolismo , Transdução de Sinais/efeitos dos fármacos
4.
Adv Exp Med Biol ; 1185: 431-436, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884650

RESUMO

Extracellular vesicles (EVs) are membranous structures released by cells, including those of the retinal pigment epithelium (RPE) and photoreceptors. The cargo of EVs includes genetic material and proteins, making these vesicles essential in cell communication. Among the genetic materials, we find a large number of microRNAs (miRNAs), small chains of noncoding RNA. In the case of EVs from the retina, changes have also been observed in the number and cargo of EVs.Our group confirmed that damaged RPE cells in vitro release a greater number of EVs with a higher pro-angiogenic factor (VEGFR-1 and VEGFR-2) than control non-damaged cells, thus increasing neovascularization in endothelial cell cultures. This indicates that something similar could happen in patients suffering from some types of retinal degeneration that occur with angiogenesis, such as wet AMD or RD.Here, we investigated the role of EVs in photoreceptor degeneration, and we report for the first time on CD9 and CD81, closely related tetraspanins, in wild-type and rd1 retinae. Our study demonstrates the involvement of EVs in the process of inherited photoreceptor degeneration in a PDE6 mutation.


Assuntos
Vesículas Extracelulares , Degeneração Retiniana/patologia , Retinose Pigmentar/patologia , Tetraspanina 28/metabolismo , Tetraspanina 29/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Humanos , Retina , Epitélio Pigmentado da Retina/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
5.
Hum Mol Genet ; 25(20): 4462-4472, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28172811

RESUMO

Cone photoreceptor cell death as it occurs in certain hereditary retinal diseases is devastating, with the affected patients suffering from a loss of accurate and colour vision. Regrettably, these hereditary cone diseases are still untreatable to date. Thus, the identification of substances able to block or restrain cone cell death is of primary importance. We studied the neuroprotective effects of a histone deacetylase inhibitor, Trichostatin A (TSA), in a mouse model of inherited, primary cone degeneration (cpfl1). We show that HDAC inhibition protects cpfl1 cones in vitro, in retinal explant cultures. More importantly, in vivo, a single intravitreal TSA injection significantly increased cone survival for up to 16 days post-injection. In addition, the abnormal, incomplete cone migration pattern in the cpfl1 retina was significantly improved by HDAC inhibition. These findings suggest a crucial role for HDAC activity in primary cone degeneration and highlight a new avenue for future therapy developments for cone dystrophies and retinal diseases associated with impaired cone migration.


Assuntos
Modelos Animais de Doenças , Ácidos Hidroxâmicos/farmacologia , Fármacos Neuroprotetores/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/tratamento farmacológico , Animais , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Camundongos , Fármacos Neuroprotetores/uso terapêutico
6.
Hum Mol Genet ; 24(19): 5486-99, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26188004

RESUMO

Mutations in the PDE6A gene can cause rod photoreceptors degeneration and the blinding disease retinitis pigmentosa (RP). While a number of pathogenic PDE6A mutations have been described, little is known about their impact on compound heterozygous situations and potential interactions of different disease-causing alleles. Here, we used a novel mouse model for the Pde6a R562W mutation in combination with an existing line carrying the V685M mutation to generate compound heterozygous Pde6a V685M/R562W animals, exactly homologous to a case of human RP. We compared the progression of photoreceptor degeneration in these compound heterozygous mice with the homozygous V685M and R562W mutants, and additionally with the D670G line that is known for a relatively mild phenotype. We investigated PDE6A expression, cyclic guanosine mono-phosphate accumulation, calpain and caspase activity, in vivo retinal function and morphology, as well as photoreceptor cell death and survival. This analysis confirms the severity of different Pde6a mutations and indicates that compound heterozygous mutants behave like intermediates of the respective homozygous situations. Specifically, the severity of the four different Pde6a situations may be categorized by the pace of photoreceptor degeneration: V685M (fastest) > V685M/R562W > R562W > D670G (slowest). While calpain activity was strongly increased in all four mutants, caspase activity was not. This points to the execution of non-apoptotic cell death and may lead to the identification of new targets for therapeutic interventions. For individual RP patients, our study may help to predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window-of-opportunity for clinical interventions.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Mutação Puntual , Retina/fisiopatologia , Retinose Pigmentar/patologia , Animais , Calpaína/metabolismo , Caspases/metabolismo , Sobrevivência Celular , Modelos Animais de Doenças , Humanos , Camundongos , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/fisiopatologia
7.
Cell Mol Life Sci ; 72(20): 3953-69, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25939269

RESUMO

The unconventional myosin VI, a member of the actin-based motor protein family of myosins, is expressed in the retina. Its deletion was previously shown to reduce amplitudes of the a- and b-waves of the electroretinogram. Analyzing wild-type and myosin VI-deficient Snell's Waltzer mice in more detail, the expression pattern of myosin VI in retinal pigment epithelium, outer limiting membrane, and outer plexiform layer could be linked with differential progressing ocular deficits. These encompassed reduced a-waves and b-waves and disturbed oscillatory potentials in the electroretinogram, photoreceptor cell death, retinal microglia infiltration, and formation of basal laminar deposits. A phenotype comprising features of glaucoma (neurodegeneration) and age-related macular degeneration could thus be uncovered that suggests dysfunction of myosin VI and its variable cargo adaptor proteins for membrane sorting and autophagy, as possible candidate mediators for both disease forms.


Assuntos
Deleção de Genes , Degeneração Macular/genética , Cadeias Pesadas de Miosina/fisiologia , Doenças do Nervo Óptico/genética , Animais , Genótipo , Degeneração Macular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Doenças do Nervo Óptico/patologia , Células Fotorreceptoras de Vertebrados/patologia , Retina/metabolismo , Retina/fisiologia
8.
J Biol Chem ; 288(50): 36129-40, 2013 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-24187136

RESUMO

Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing (13)C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia.


Assuntos
Ácido Aspártico/metabolismo , Ácido Glutâmico/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Purinonas/farmacologia , Ácido Pirúvico/metabolismo , Retina/citologia , Animais , Transporte Biológico/efeitos dos fármacos , Encéfalo/citologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Metabolômica , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oxigênio/metabolismo
9.
Adv Exp Med Biol ; 801: 463-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24664732

RESUMO

Poly(ADP-ribose) (PAR) turnover is required for many cellular processes, and highly relevant for cell death and survival. This post-translational protein modification is regulated by the synthesizing enzyme poly(ADP)ribose-polymerase (PARP) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). Previously, PARP activity was found to be involved in photoreceptor degeneration in the rd1 mouse and in rd1-like conditions PARP-1 was the main PARP family member contributing to photoreceptor cell death. Despite the manifest role of PARP and PAR accumulation in photoreceptor cell death, the influence of PAR degradation on photoreceptor viability was still unknown. Here, we investigated the role of PARG in photoreceptor degeneration using the PARG-110 knock out mouse and report for the first time on PARG expression in wild-type and knock-out retina.


Assuntos
Glicosídeo Hidrolases/genética , Poli(ADP-Ribose) Polimerases/genética , Retina/fisiologia , Degeneração Retiniana/fisiopatologia , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Camundongos , Camundongos Knockout , Fármacos Neuroprotetores/farmacologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/fisiologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Retina/efeitos dos fármacos , Retina/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo
10.
Sci Rep ; 9(1): 3758, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842506

RESUMO

Retinitis Pigmentosa is a group of inherited neurodegenerative diseases that result in selective cell death of photoreceptors. In the developed world, RP is regarded as the main cause of blindness among the working age population. The precise mechanisms eventually leading to cell death remain unknown and to date no adequate treatment for RP is available. Poly ADP ribose polymerase (PARP) over activity is involved in photoreceptor degeneration and pharmacological inhibition or genetic knock-down PARP1 activity protect photoreceptors in mice models, the mechanism of neuroprotection is not clear yet. Our result indicated that olaparib, a PARP1 inhibitor, significantly rescued photoreceptor cells in rd10 retina. Extracellular vesicles (EVs) were previously recognized as a mechanism for discharging useless cellular components. Growing evidence has elucidated their roles in cell-cell communication by carrying nucleic acids, proteins and lipids that can, in turn, regulate behavior of the target cells. Recent research suggested that EVs extensively participate in progression of diverse blinding diseases, such as age-related macular (AMD) degeneration. Our study demonstrates the involvement of EVs activity in the process of photoreceptor degeneration in a PDE6 mutation. PARP inhibition protects photoreceptors via regulation of the EVs activity in rod photoreceptor degeneration in a PDE6b mutation.


Assuntos
Células Fotorreceptoras de Vertebrados/citologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Retinose Pigmentar/patologia , Animais , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Humanos , Camundongos , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Poli ADP Ribosilação , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo
11.
PLoS One ; 12(7): e0181374, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28723922

RESUMO

Peripherin (peripherin/rds) is a membrane-associated protein that plays a critical role in the morphogenesis of rod and cone photoreceptor outer segments. Mutations in the corresponding PRPH2 gene cause different types of retinal dystrophies characterized by a loss of photoreceptors. Over activation of poly-ADP-ribose polymerase (PARP) was previously shown to be involved in different animal models for hereditary retinal dystrophies. This includes the rd2 mouse, which suffers from a human homologous mutation in the PRPH2 gene. In the present study, we show that increased retinal PARP activity and poly-ADP-ribosylation of proteins occurs before the peak of rd2 photoreceptor degeneration. Inhibition of PARP activity with the well-characterized PARP inhibitor PJ34 decreased the levels of poly-ADP-ribosylation and photoreceptor cell death. These results suggest a causal involvement of PARP in photoreceptor degeneration caused by peripherin mutations and highlight the possibility to use PARP inhibition for the mutation-independent treatment of hereditary retinal dystrophies.


Assuntos
Fármacos Neuroprotetores/farmacologia , Periferinas/metabolismo , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Degeneração Retiniana/prevenção & controle , Animais , Camundongos , Camundongos Knockout , Periferinas/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/metabolismo
12.
ALTEX ; 33(4): 459-464, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27159027

RESUMO

Diabetic retinopathy (DR) is a major cause of vision loss and one of the most common and debilitating complications of diabetes. Research to prevent DR is hindered by a lack of experimental model systems that faithfully reproduce the disease pathology, in particular for type 2 diabetes, which requires prolonged disease progression in animals to develop some hallmarks of DR. Here, we introduce an alternative in vitro model system for DR, based on serum-free, organotypic rodent retinal explant cultures, which allow physiological and pharmacological manipulation of the retina for up to two weeks under tightly controlled conditions. Retinal explant cultures have the advantage of isolating direct neuronal consequences of diabetic conditions from indirect systemic effects mediated via the retinal vasculature or the immune system. Exposed to conditions emulating type 1 or type 2 diabetes, retinal explants displayed elevated cell death rates among inner retinal neurons as well as photoreceptors, with a particularly strong loss of cone photoreceptors. Our results support a direct impact of diabetic conditions on retinal neurons and may help explain color vision defects observed in DR patients. This serum-free in vitro DR model avoids the animal suffering of established DR models and reduces the overall number of animals needed for such research. It should prove useful to study the mechanisms of neuronal cell death caused by DR and to screen for potential future DR treatments.


Assuntos
Retinopatia Diabética/induzido quimicamente , Glucose/toxicidade , Insulina/toxicidade , Retina/efeitos dos fármacos , Técnicas de Cultura de Tecidos/métodos , Animais , Caspase 3/genética , Caspase 3/metabolismo , Ativação Enzimática , Camundongos Endogâmicos C3H , Células Fotorreceptoras Retinianas Cones
13.
Sci Rep ; 6: 39537, 2016 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-28004814

RESUMO

The enzyme poly-ADP-ribose-polymerase (PARP) mediates DNA-repair and rearrangements of the nuclear chromatin. Generally, PARP activity is thought to promote cell survival and in recent years a number of PARP inhibitors have been clinically developed for cancer treatment. Paradoxically, PARP activity is also connected to many diseases including the untreatable blinding disease Retinitis Pigmentosa (RP), where PARP activity appears to drive the pathogenesis of photoreceptor loss. We tested the efficacy of three different PARP inhibitors to prevent photoreceptor loss in the rd1 mouse model for RP. In retinal explant cultures in vitro, olaparib had strong and long-lasting photoreceptor neuroprotective capacities. We demonstrated target engagement by showing that olaparib reduced photoreceptor accumulation of poly-ADP-ribosylated proteins. Remarkably, olaparib also reduced accumulation of cyclic-guanosine-monophosphate (cGMP), a characteristic marker for photoreceptor degeneration. Moreover, intravitreal injection of olaparib in rd1 animals diminished PARP activity and increased photoreceptor survival, confirming in vivo neuroprotection. This study affirms the role of PARP in inherited retinal degeneration and for the first time shows that a clinically approved PARP inhibitor can prevent photoreceptor degeneration in an RP model. The wealth of human clinical data available for olaparib highlights its strong potential for a rapid clinical translation into a novel RP treatment.


Assuntos
Fármacos Neuroprotetores/farmacologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/genética , Animais , Sobrevivência Celular , Cromatina/metabolismo , GMP Cíclico/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C3H , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Coelhos , Degeneração Retiniana/patologia
14.
PLoS One ; 9(11): e112142, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25392995

RESUMO

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.


Assuntos
Morte Celular/fisiologia , Degeneração Retiniana/fisiopatologia , Animais , Animais Geneticamente Modificados , Calpaína/fisiologia , Morte Celular/genética , GMP Cíclico/fisiologia , Modelos Animais de Doenças , Histona Desacetilases/fisiologia , Transdução de Sinal Luminoso/genética , Camundongos , Mutação , Poli Adenosina Difosfato Ribose/fisiologia , Poli(ADP-Ribose) Polimerases/fisiologia , Ratos , Degeneração Retiniana/genética
15.
PLoS One ; 6(7): e22181, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21765948

RESUMO

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.


Assuntos
Calpaína/metabolismo , Mutação/genética , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Rodopsina/genética , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Morte Celular , Forma Celular , Citocromos c/metabolismo , Dano ao DNA , Ativação Enzimática , Humanos , Marcação In Situ das Extremidades Cortadas , Estresse Oxidativo , Poli Adenosina Difosfato Ribose/metabolismo , Transporte Proteico , Ratos , Ratos Mutantes , Ratos Transgênicos , Coloração e Rotulagem
16.
PLoS One ; 5(11): e15495, 2010 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-21124852

RESUMO

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.


Assuntos
Células Fotorreceptoras de Vertebrados/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Retina/enzimologia , Degeneração Retiniana/enzimologia , Animais , Apoptose , Western Blotting , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/antagonistas & inibidores , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Eletrorretinografia , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C3H , Camundongos Knockout , Inibidores de Fosfodiesterase/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Purinonas/farmacologia , Retina/metabolismo , Retina/fisiopatologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Retinose Pigmentar/enzimologia , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Tomografia de Coerência Óptica
17.
Graefes Arch Clin Exp Ophthalmol ; 244(5): 627-33, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16205934

RESUMO

BACKGROUND: Oxidative stress during ischemia-reperfusion (I/R) is thought to be a major cause of retinal injury after I/R. The present study was aimed at investigating the protective role of antioxidant application. METHODS: Four commonly used antioxidants (vitamin E=alpha tocopherol, lutein, fenugreek=Trigonella foenum-graecum and germander=Teucrium multicaule) were applied in ischemia-reperfusion (I/R) injury of the right retinae of 51 adult pigmented rats. Each of the antioxidants was administered every 6 h, beginning 6 h before the ischemia. After 60 min ischemia and 24 h reperfusion, we assayed (1) oxidative damage by measuring malondialdehyde (MDA), (2) apoptosis by measuring activated caspase-3 (using immunoblots), and (3) intrinsic antioxidative capacity by measuring glutathione (GSH) levels in the retinae. RESULTS: In the order of lutein>Trigonella>vitamin E>Teucrium, all four compounds were effective in preventing retinal damage by I/R, as (1) they significantly decreased the formation of MDA (8.83, 16.48, 17.24, 18.5 nmol/100 mg tissue wet weight, respectively) compared with I/R without protection (23.29 nmol/100 mg tissue wet weight; controls: 8.0 nmol/100 mg tissue wet weight); (2) they significantly inhibited the activation of caspase-3 [0.01, 0.02, 0,02, and 0.04 arbitrary units (AU), respectively, versus control, 0.0, and I/R, 0.08 AU]; and (3) they significantly decelerated the loss of GSH (from control levels of 36.04 nmol/100 mg tissue wet weight) to 30.4, 15.98, 18.1, 15.02 nmol/100 mg tissue wet weight, respectively (lutein, Trigonella, vitamin E, Teucrium), compared with unprotected I/R (12.84 nmol/100 mg tissue wet weight). CONCLUSIONS: Our study shows that lutein, Trigonella, Teucrium and vitamin E exert protection against in vivo retinal I/R injury in rats; this may recommend these compounds (in particular, lutein) for clinical use in patients with different types of ocular I/R injuries.


Assuntos
Antioxidantes/administração & dosagem , Traumatismo por Reperfusão/prevenção & controle , Doenças Retinianas/prevenção & controle , Vasos Retinianos/efeitos dos fármacos , Animais , Apoptose , Caspase 3/metabolismo , Modelos Animais de Doenças , Glutationa/metabolismo , Luteína/administração & dosagem , Masculino , Malondialdeído/metabolismo , Estresse Oxidativo , Fitoterapia , Preparações de Plantas/administração & dosagem , Ratos , Ratos Long-Evans , Traumatismo por Reperfusão/metabolismo , Retina/metabolismo , Doenças Retinianas/metabolismo , Vasos Retinianos/metabolismo , Teucrium , Trigonella , Vitamina E/administração & dosagem
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