RESUMO
BACKGROUND: Traumatic brain injury (TBI) is associated with the development of visual system disorders. Visual deficits can present with delay and worsen over time, and may be associated with an ongoing neuroinflammatory response that is known to occur after TBI. Complement system activation is strongly associated with the neuroinflammatory response after TBI, but whether it contributes to vision loss after TBI is unexplored. METHODS: Acute and chronic neuroinflammatory changes within the dorsal lateral geniculate nucleus (dLGN) and retina were investigated subsequent to a moderate to severe murine unilateral controlled cortical impact. Neuroinflammatory and histopathological outcomes were interpreted in the context of behavioral and visual function data. To investigate the role of complement, cohorts were treated after TBI with the complement inhibitor, CR2-Crry. RESULTS: At 3 days after TBI, complement component C3 was deposited on retinogeniculate synapses in the dLGN both ipsilateral and contralateral to the lesion, which was reduced in CR2-Crry treated animals. This was associated with microglia morphological changes in both the ipsilateral and contralateral dLGN, with a less ramified phenotype in vehicle compared to CR2-Crry treated animals. Microglia in vehicle treated animals also had a greater internalized VGlut2 + synaptic volume after TBI compared to CR2-Crry treated animals. Microglia morphological changes seen acutely persisted for at least 49 days after injury. Complement inhibition also reduced microglial synaptic internalization in the contralateral dLGN and increased the association between VGLUT2 and PSD95 puncta, indicating preservation of intact synapses. Unexpectedly, there were no changes in the thickness of the inner retina, retinal nerve fiber layer or retinal ganglion layer. Neuropathological changes in the dLGN were accompanied by reduced visual acuity at subacute and chronic time points after TBI, with improvement seen in CR2-Crry treated animals. CONCLUSION: TBI induces complement activation within the dLGN and promotes microglial activation and synaptic internalization. Complement inhibition after TBI in a clinically relevant paradigm reduces complement activation, maintains a more surveillance-like microglia phenotype, and preserves synaptic density within the dLGN. Together, the data indicate that complement plays a key role in the development of visual deficits after TBI via complement-dependent microglial phagocytosis of synapses within the dLGN.
Assuntos
Lesões Encefálicas Traumáticas , Animais , Camundongos , Lesões Encefálicas Traumáticas/patologia , Complemento C3/genética , Ativação do Complemento , Células Ganglionares da Retina/patologia , Inflamação/complicações , Proteínas Recombinantes de FusãoRESUMO
PURPOSE: Recent reports linking HDAC6 to mitochondrial turnover and neurodegeneration led us to hypothesize that an inhibitor such as Vorinostat (suberoylanilide hydroxamic acid, SAHA) may reduce mitochondrial damage found in retinitis pigmentosa (RP), a progressive neurodegenerative disease of the eye. Here we tested the efficacy of SAHA for its ability to protect photoreceptors in in-vitro and in-situ models of RP. As the stressor, we focused on calcium overload. Calcium is one of the main drivers of cell death, and is associated with rod loss in the rd1 mouse retina, which harbors a mutation in the Pde6b gene similar to that found in human patients suffering from autosomal recessive RP. METHOD: Murine photoreceptor cell line (661W) were exposed to agents that led to calcium stress. Cell survival and redox capacity were measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, real-time changes in cellular metabolism were assessed using the Seahorse Biosciences XF24 analyzer, and mitochondrial fission-fusion using imaging. In-situ, neuroprotection was assessed in RPE/retina organ cultures of the rd1 mouse. SAHA effects on cell survival were compared in 661W cells with those of the specific HDAC6 inhibitor tubastatin A, and those on protein acetylation by Western blotting. RESULTS: In stressed 661W cells, SAHA was found to increase cell survival that was associated with improved mitochondrial respiration and reduced mitochondrial fission. The protective effects of SAHA were also observed on photoreceptor cell survival in whole retinal organ explants of the rd1 mouse. Even though tubastatin A was ineffective in increasing cell survival in 661W cells, HDAC6 activity was confirmed in 661W cells after SAHA treatment with protein acetylation specific for HDAC6, defined by an increase in tubulin, but not histone acetylation. CONCLUSIONS: SAHA was found to protect mitochondria from damage, and concomitantly reduced photoreceptor cell death in cell and organ cultures. The lack of activity of tubastatin A suggests that there must be an additional mechanism of action involved in the protective mechanism of SAHA that is responsible for its neuroprotection. Overall, SAHA may be a useful treatment for the prevention of photoreceptor degeneration associated with human RP. The results are discussed in the context of the effects of inhibitors that target different classes and members of the HDAC family and their effects on rod versus cone survival.
Assuntos
Modelos Animais de Doenças , Inibidores de Histona Desacetilases/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Retinose Pigmentar/tratamento farmacológico , Vorinostat/uso terapêutico , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Doenças Mitocondriais/prevenção & controle , NADH NADPH Oxirredutases/metabolismo , Técnicas de Cultura de Órgãos , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologiaRESUMO
Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with multiple sclerosis. ON pathology is characterized by attack of autoreactive T cells against optic nerve antigens, resulting in demyelination, death of retinal ganglion cells, and cumulative visual impairment. A model of experimental autoimmune encephalomyelitis (EAE) was utilized to study the onset and progression of ON and the neuroprotective efficacy of oral treatment with the calpain inhibitor SNJ 1945. EAE was actively induced in B10.PL mice with myelin basic protein on Days 0 and 2, and mice received twice daily oral dosing of SNJ 1945 from Day 9 until sacrificing (Day 26). Visual function was determined by electroretinogram recordings and daily measurement of optokinetic responses (OKR) to a changing pattern stimulus. Optic nerve and retinal histopathology was investigated by immunohistochemical and luxol fast blue staining. EAE mice manifested losses in OKR thresholds, a measurement of visual acuity, which began early in the disease course. There was a significant bias toward unilateral OKR impairment among EAE-ON eyes. Treatment with SNJ 1945, initiated after the onset of OKR threshold decline, improved visual acuity, pattern electroretinogram amplitudes, and paralysis, with attenuation of retinal ganglion cell death. Furthermore, calpain inhibition spared oligodendrocytes, prevented degradation of axonal neurofilament protein, and attenuated reactive astrocytosis. The trend of early, unilateral visual impairment in EAE-ON parallels the clinical presentation of ON exacerbations associated with multiple sclerosis. Calpain inhibition may represent an ideal candidate therapy for the preservation of vision in clinical ON. As in multiple sclerosis (MS) patients, optic neuritis (ON) and early, primarily monocular loss in spatial acuity is observed in a rodent model (EAE, experimental autoimmune encephalomyelitis). Daily oral treatment with the calpain inhibitor SNJ 1945 preserves visual acuity and preserves retinal ganglion cells (Brn3a, brain-specific homeobox/POU domain protein 3A) and their axons (MOSP, myelin oligodendrocyte-specific protein). Calpain inhibition may represent a candidate therapy for the preservation of vision in ON.
Assuntos
Calpaína/antagonistas & inibidores , Carbamatos/farmacologia , Carbamatos/uso terapêutico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Neurite Óptica/tratamento farmacológico , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Eletrorretinografia/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Gliose/prevenção & controle , Masculino , Camundongos , Proteína Básica da Mielina/metabolismo , Nistagmo Optocinético/efeitos dos fármacos , Neurite Óptica/etiologia , Neurite Óptica/fisiopatologia , Estimulação Luminosa , Acuidade Visual/efeitos dos fármacosRESUMO
Background: Traumatic brain injury (TBI) is associated with the development of visual system disorders. Visual deficits can present with delay and worsen over time, and may be associated with an ongoing neuroinflammatory response that is known to occur after TBI. Complement activation is strongly associated with the neuroinflammatory response after TBI, but whether it contributes to vision loss after TBI is unexplored. Methods: Acute and chronic neuroinflammatory changes within the dorsal lateral geniculate nucleus (dLGN) and retina were investigated subsequent to murine controlled unilateral cortical impact. Neuroinflammatory and histopathological data were interpreted in the context of behavioral and visual function data. To investigate the role of complement, cohorts were treated after TBI with the complement inhibitor, CR2-Crry. Results: At 3 days after TBI, complement C3 was deposited on retinogeniculate synapses in the dLGN both ipsilateral and contralateral to the lesion, which was reduced in CR2-Crry treated animals. This was associated with microglia morphological changes in both the ipsilateral and contralateral dLGN, with a more amoeboid phenotype in vehicle compared to CR2-Crry treated animals. Microglia in vehicle treated animals also had a greater internalized VGlut2+ synaptic volume after TBI compared to CR2-Crry treated animals. Microglia morphological changes seen acutely persisted for at least 49 days after injury. Complement inhibition also reduced microglial synaptic internalization in the contralateral dLGN and increased the association between VGLUT2 and PSD95 puncta, indicating preservation of intact synapses. Unexpectedly, there were no changes in the thickness of the inner retina, retinal nerve fiber layer or retinal ganglion layer. Pathologies were accompanied by reduced visual acuity at subacute and chronic time points after TBI, with improvement seen in CR2-Crry treated animals. Conclusion: TBI induces complement activation within the dLGN and promotes microglial activation and synaptic internalization. Complement inhibition after TBI in a clinically relevant paradigm reduces complement activation, maintains a more surveillance-like microglia phenotype, and preserves synaptic density within the dLGN. Together, the data indicate that complement plays a key role in the development of visual deficits after TBI via complement-dependent microglial phagocytosis of synapses within the dLGN.
RESUMO
Rpe65(-/-) mice are unable to produce 11-cis-retinal, the chromophore of visual pigments. Consequently, the pigment is present as the apoprotein opsin with a minute level of pigment containing 9-cis-retinal as chromophore. Notably, a 10-20% fraction of this opsin is mono-phosphorylated independently of light conditions. To determine the role of rhodopsin kinase (GRK1) in phosphorylating this opsin and to test whether eliminating this phosphorylation would accelerate photoreceptor degeneration, we generated the Rpe65(-/-)Grk1(-/-) mouse. The retinae of Rpe65(-/-)Grk1(-/-) mice had negligible opsin phosphorylation, extensive degeneration with decreased opsin levels, and diminished light-evoked rod responses relative to Rpe65(-/-) mice. These data show that opsin phosphorylation in the Rpe65(-/-) mouse is due to the action of GRK1 and is neuroprotective. However, despite the higher activity of unphosphorylated opsin, the severe loss of opsin in the rapidly degenerating Rpe65(-/-)Grk1(-/-) mice resulted in lower overall opsin activity and in higher rod sensitivity compared with Rpe65(-/-) mice. In Rpe65(-/-)Grk1(-/-)Gnat1(-/-) mice where transduction activation was blocked, degeneration was only partially prevented. Therefore, increased opsin activity in the absence of phosphorylation was not the only mechanism for the accelerated retinal degeneration. Finally, the deletion of GRK1 triggered retinal degeneration in Grk1(-/-) mice after 1 month, even in the absence of apo-opsin. This degeneration was independent of light conditions and occurred even in the absence of transducin in Grk1(-/-)Gnat1(-/-) mice. Taken together, our results demonstrate a light-independent mechanism for retinal degeneration in the absence of GRK1, suggesting a second, not previously recognized role for that kinase.
Assuntos
Receptor Quinase 1 Acoplada a Proteína G/deficiência , Degeneração Retiniana/genética , Transducina/metabolismo , Visão Ocular/genética , Adaptação Ocular/genética , Animais , Biofísica/métodos , Proteínas de Transporte/genética , Proteínas do Olho/genética , Subunidades alfa de Proteínas de Ligação ao GTP/deficiência , Camundongos , Camundongos Knockout , Opsinas/metabolismo , Fosforilação/genética , Estimulação Luminosa/métodos , Degeneração Retiniana/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , cis-trans-IsomerasesRESUMO
MOTIVATION: The results of initial analyses for many high-throughput technologies commonly take the form of gene or protein sets, and one of the ensuing tasks is to evaluate the functional coherence of these sets. The study of gene set function most commonly makes use of controlled vocabulary in the form of ontology annotations. For a given gene set, the statistical significance of observing these annotations or 'enrichment' may be tested using a number of methods. Instead of testing for significance of individual terms, this study is concerned with the task of assessing the global functional coherence of gene sets, for which novel metrics and statistical methods have been devised. RESULTS: The metrics of this study are based on the topological properties of graphs comprised of genes and their Gene Ontology annotations. A novel aspect of these methods is that both the enrichment of annotations and the relationships among annotations are considered when determining the significance of functional coherence. We applied our methods to perform analyses on an existing database and on microarray experimental results. Here, we demonstrated that our approach is highly discriminative in terms of differentiating coherent gene sets from random ones and that it provides biologically sensible evaluations in microarray analysis. We further used examples to show the utility of graph visualization as a tool for studying the functional coherence of gene sets. AVAILABILITY: The implementation is provided as a freely accessible web application at: http://projects.dbbe.musc.edu/gosteiner. Additionally, the source code written in the Python programming language, is available under the General Public License of the Free Software Foundation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Biologia Computacional/métodos , Genes , Animais , Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Humanos , SoftwareRESUMO
Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models, reasoning drugs effective across species and/or independent of disease mutation may translate better clinically. We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP. We tested 2934 compounds, mostly human-approved drugs, across six concentrations, resulting in 113 compounds being identified as hits. Secondary tests of 42 high-priority hits confirmed eleven lead candidates. Leads were then evaluated in a series of mouse RP models in an effort to identify compounds effective across species and RP models, that is, potential pan-disease therapeutics. Nine of 11 leads exhibited neuroprotective effects in mouse primary photoreceptor cultures, and three promoted photoreceptor survival in mouse rd1 retinal explants. Both shared and complementary mechanisms of action were implicated across leads. Shared target tests implicated parp1-dependent cell death in our zebrafish RP model. Complementation tests revealed enhanced and additive/synergistic neuroprotective effects of paired drug combinations in mouse photoreceptor cultures and zebrafish, respectively. These results highlight the value of cross-species/multi-model phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for RP patients.
Photoreceptors are the cells responsible for vision. They are part of the retina: the light-sensing tissue at the back of the eye. They come in two types: rods and cones. Rods specialise in night vision, while cones specialise in daytime colour vision. The death of these cells can cause a disease, called retinitis pigmentosa, that leads to vision loss. Symptoms often start in childhood with a gradual loss of night vision. Later on, loss of cone photoreceptors can lead to total blindness. Unfortunately, there are no treatments available that protect photoreceptor cells from dying. Research has identified drugs that can protect photoreceptors in animal models, but these drugs have failed in humans. The classic way to look for new treatments is to find drugs that target molecules implicated in a disease, and then test them to see if they are effective. Unfortunately, many drugs identified in this way fail in later stages of testing, either because they are ineffective, or because they have unacceptable side effects. One way to reverse this trend is to first test whether a drug is effective at curing a disease in animals, and later determining what it does at a molecular level. This could reveal whether drugs can protect photoreceptors before research to discover their molecular targets begins. Tests like this across different species could maximise the chances of finding a drug that works in humans, because if a drug works in several species, it is more likely to have shared target molecules across species. Applying this reasoning, Zhang et al. tested around 3,000 drug candidates for treating retinitis pigmentosa in a strain of zebrafish that undergoes photoreceptor degeneration similar to the human disease. Most of these drug candidates already have approval for use in humans, meaning that if they were found to be effective for treating retinitis pigmentosa, they could be fast-tracked for use in people. Zhang et al. found three compounds that helped photoreceptors survive both in zebrafish and in retinas grown in the laboratory derived from a mouse strain with degeneration similar to retinitis pigmentosa. Tests to find out how these three compounds worked at the molecular level revealed that they interfered with a protein that can trigger cell death. The tests also found other promising compounds, many of which offered increased protection when combined in pairs. Worldwide there are between 1.5 and 2.5 million people with retinitis pigmentosa. With this disease, loss of vision happens slowly, so identifying drugs that could slow or stop the process could help many people. These results suggest that placing animal testing earlier in the drug discovery process could complement traditional target-based methods. The compounds identified here, and the information about how they work, could expand potential treatment research. The next step in this research is to test whether the drugs identified by Zhang et al. protect mammals other than mice from the degeneration seen in retinitis pigmentosa.
Assuntos
Fármacos Neuroprotetores/farmacologia , Retinose Pigmentar/tratamento farmacológico , Animais , Animais Geneticamente Modificados , Células Cultivadas/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Mutação , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Peixe-ZebraRESUMO
The focus of this review is the role of complement-mediated phagocytosis in retinal and neurological diseases affecting the visual system. Complement activation products opsonize synaptic material on neurons for phagocytic removal, which is a normal physiological process during development, but a pathological process in several neurodegenerative diseases and conditions. We discuss the role of complement in the refinement and elimination of synapses in the retina and lateral geniculate nucleus, both during development and in disease states. How complement and aberrant phagocytosis promotes injury to the visual system is discussed primarily in the context of multiple sclerosis, where it has been extensively studied, although the role of complement in visual dysfunction in other diseases such as stroke and traumatic brain injury is also highlighted. Retinal diseases are also covered, with a focus on glaucoma and age-related macular degeneration. Finally, we discuss the potential of complement inhibitory strategies to treat diseases affecting the visual system.
Assuntos
Proteínas do Sistema Complemento/imunologia , Microglia/imunologia , Doenças do Sistema Nervoso/imunologia , Doenças Retinianas/imunologia , Animais , Inativadores do Complemento/uso terapêutico , Humanos , Doenças do Sistema Nervoso/tratamento farmacológico , Fagocitose , Retina/imunologia , Doenças Retinianas/tratamento farmacológicoRESUMO
Lecithin retinol acyl transferase (LRAT) and retinal pigment epithelium protein 65 (RPE65) are key enzymes of the retinoid cycle. In Lrat(-/-) and Rpe65(-/-) mice, models of human Leber congenital amaurosis, the retinoid cycle is disrupted and 11-cis-retinal, the chromophore of visual pigments, is not produced. The Lrat(-/-) and Rpe65(-/-) retina phenotype presents with rapid sectorial cone degeneration, and the visual pigments, S-opsin and M/L-opsin, fail to traffic to cone outer segments appropriately. In contrast, rod opsin traffics normally in mutant rods. Concomitantly, guanylate cyclase 1, cone T alpha-subunit, cone phosphodiesterase 6alpha' (PDE6alpha'), and GRK1 (G-protein-coupled receptor kinase 1; opsin kinase) are not transported to Lrat(-/-) and Rpe65(-/-) cone outer segments. Aberrant localization of these membrane-associated proteins was evident at postnatal day 15, before the onset of ventral and central cone degeneration. Protein levels of cone T alpha and cone PDE6alpha' were reduced, whereas their transcript levels were unchanged, suggesting posttranslational degradation. In an Rpe65(-/-)Rho(-/-) double knock-out model, trafficking of cone pigments and membrane-associated cone phototransduction polypeptides to the outer segments proceeded normally after 11-cis-retinal administration. These results suggest that ventral and central cone opsins must be regenerated with 11-cis-retinal to permit transport to the outer segments. Furthermore, the presence of 11-cis-retinal is essential for proper transport of several membrane-associated cone phototransduction polypeptides in these cones.
Assuntos
Proteínas de Membrana/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Retinaldeído/metabolismo , Aciltransferases/deficiência , Aciltransferases/metabolismo , Animais , Cegueira/congênito , Cegueira/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Guanilato Ciclase/metabolismo , Camundongos , Camundongos Knockout , Degeneração Neural/etiologia , Degeneração Neural/fisiopatologia , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiopatologia , Pigmentos da Retina/metabolismo , Retinaldeído/deficiência , Retinaldeído/farmacologia , Opsinas de Bastonetes/metabolismo , Fatores de Tempo , Visão Ocular , cis-trans-IsomerasesRESUMO
Given the established importance of glial cell line-derived neurotrophic factor (GDNF) in maintaining dopaminergic neurotransmitter systems, the nigrostriatal system and associated behaviors of mice with genetic reduction of its high-affinity receptor, GDNF receptor (GFR)alpha-1 (GFRalpha-1(+/-)), were compared with wild-type controls. Motor activity and the stimulatory effects of a dopamine (DA) D1 receptor agonist (SKF 82958) were assessed longitudinally at 8 and 18 months of age. Monoamine concentrations and dopaminergic nerve terminals in the striatum and the number of dopaminergic neurons in the substantia nigra (SN) were assessed. The results support the importance of GFRalpha-1 in maintaining normal function of the nigrostriatal dopaminergic system, with deficits being observed for GFRalpha-1(+/-) mice at both ages. Motor activity was lower and the stimulatory effects of the DA agonist were enhanced for the older GFRalpha-1(+/-) mice. DA in the striatum was reduced in the GFRalpha-1(+/-) mice at both ages, and tyrosine hydroxylase-positive cell numbers in the SN were reduced most substantially in the older GFRalpha-1(+/-) mice. The combined behavioral, pharmacological probe, neurochemical and morphological measures provide evidence of abnormalities in GFRalpha-1(+/-) mice that are indicative of an exacerbated aging-related decline in dopaminergic system function. The noted deficiencies, in turn, suggest that GFRalpha-1 is necessary for GDNF to maintain normal function of the nigrostriatal dopaminergic system. Although the precise mechanism(s) for the aging-related changes in the dopaminergic system remain to be established, the present study clearly establishes that genetic reductions in GFRalpha-1 can contribute to the degenerative changes observed in this system during the aging process.
Assuntos
Envelhecimento/metabolismo , Corpo Estriado/metabolismo , Dopamina/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Substância Negra/metabolismo , Envelhecimento/genética , Animais , Corpo Estriado/citologia , Agonistas de Dopamina/farmacologia , Heterozigoto , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Vias Neurais/citologia , Vias Neurais/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Substância Negra/citologiaRESUMO
PURPOSE: The Rpe65-/- mouse, used as a model for Leber congenital amaurosis, has slow rod degeneration and rapid cone loss, presumably because of the mistrafficking of cone opsins. This animal does not generate 11-cis retinal, and both cone loss and rod response are restored by 11-cis retinal administration. Similarly, the Lrat-/- mouse does not produce 11-cis retinal. The authors sought to determine whether the same effects on rod and cone opsins in the Rpe65-/- mouse are also present in the Lrat-/- mouse, thereby establishing that these changes can be attributed to the lack of 11-cis retinal rather than to some unknown function of RPE65. METHODS: Rod and cone opsins were localized by immunohistochemical methods. Functional opsin levels were determined by regeneration with 11-cis retinal. Isorhodopsin levels were determined from pigment extraction. Opsin phosphorylation was determined by mass spectrometry. RESULTS: Rods in both models degenerated slowly. Regenerable rod opsin levels were similar over the 6-month time course investigated, rod opsin was phosphorylated at a low level (approximately 10%), and minimal 9-cis retinal was generated by a nonphotic process, giving a trace light response. In both models, S-opsin and M/L-opsin failed to traffic to the cone outer segments appropriately, and rapid cone degeneration occurred. Cone opsin mistrafficking in both models was arrested on 11-cis retinal administration. CONCLUSIONS: These data show that the Lrat-/- and Rpe65-/- mice are comparable models for studies of Leber congenital amaurosis and that the destructive cone opsin mistrafficking is caused by the lack of 11-cis retinal.
Assuntos
Aciltransferases/fisiologia , Cegueira/metabolismo , Proteínas de Transporte/fisiologia , Modelos Animais de Doenças , Proteínas do Olho/fisiologia , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneração Retiniana/metabolismo , Animais , Cegueira/congênito , Cegueira/patologia , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Camundongos Knockout , Fosforilação , Degeneração Retiniana/congênito , Degeneração Retiniana/patologia , Retinaldeído/administração & dosagem , Retinaldeído/deficiência , Opsinas de Bastonetes/metabolismo , cis-trans-IsomerasesRESUMO
Macular degeneration is hallmarked by retinal accumulation of toxic retinoid species (e.g., A2E) for which there is no endogenous mechanism to eliminate it. This ultimately results in progressive dysfunction and loss of vision either in advanced age for genetically normal patients (age-related macular degeneration) or in adolescence for those with inherited genetic mutations (Stargardt's disease). In this article, we present a proof-of-concept study for an enzyme-based therapy to remove these retinoids, modeled on traditional enzyme replacement therapy. Recombinant manganese peroxidase (rMnP) is produced in Pichia pastoris. In vitro, we demonstrate that rMnP breaks down A2E and other lipofuscin fluorophores with limited cellular toxicity, and as this enzyme is mannosylated, it can be taken up into cells through mannose receptor-dependent endocytosis. In vivo, we demonstrate that rMnP can significantly reduce the A2E burden when administered by intravitreal injections. Together, these data provide encouraging results toward the development of an enzyme-based therapy for macular degeneration and indicate the need for additional work to characterize the molecular mechanism of A2E breakdown and to improve the pharmacological parameters of the enzyme.
Assuntos
Modelos Animais de Doenças , Degeneração Macular/congênito , Degeneração Macular/terapia , Peroxidases/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Retinoides/metabolismo , Transportadores de Cassetes de Ligação de ATP/fisiologia , Animais , Células Cultivadas , Humanos , Lipofuscina/metabolismo , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos , Camundongos Knockout , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Doença de StargardtRESUMO
PURPOSE: Experimental autoimmune uveitis (EAU), an animal model of human uveitis, is an organ-specific autoimmune disease mediated by various inflammatory cytokines. In particular, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and interferon (IFN)-gamma are known to play a role in its pathogenesis. S-nitrosothiol S-nitrosoglutathione (GSNO), a slow nitric oxide (NO) donor, was reported to have beneficial effects in inflammatory disease in ischemia-reperfusion injury. The efficacy of GSNO treatment on interphotoreceptor retinoid-binding protein (IRBP)-induced EAU was investigated, using functional, histologic, and immunologic readouts. METHODS: Mice were immunized with a single injection of IRBP(161180) peptide to induce EAU, followed by a daily treatment with GSNO (1 mg/kg). Electroretinogram (ERG) analysis, histopathology, and immunologic responses to IRBP were analyzed. The effects of GSNO treatment on the antigen-specific T-cell recall responses and their cytokine production were determined. RESULTS: A single immunization of IRBP(161180) peptide led to significant structural damage of the retina and concomitant elimination of ERGs. Daily oral GSNO treatment from days 1-14 following immunization was found to be effective against IRBP-induced EAU. Histopathologic and ERG analysis both demonstrated significant retinal protection in GSNO-treated mice. The GSNO treatment of EAU animals significantly attenuated the levels of TNF-alpha, IL-1beta, IFN-gamma, and IL-10 in retinas, as measured by quantitative real-time polymerase chain reaction analysis. The splenocytes isolated from EAU- and GSNO-treated mice had lower antigen-specific T-cell proliferation in response to IRBP protein, and their cytokine production was inhibited. CONCLUSIONS: The oral administration of GSNO significantly suppressed the levels of inflammatory mediators in the retinas of EAU mice. This suppression was associated with the maintenance of normal retinal histology and function. These results clearly demonstrated the therapeutic potential of GSNO in EAU, and provide new insights for the treatment of human uveitis.
Assuntos
Doenças Autoimunes/prevenção & controle , Doadores de Óxido Nítrico/farmacologia , S-Nitrosoglutationa/farmacologia , Uveíte/prevenção & controle , Administração Oral , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/fisiopatologia , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho , Masculino , Camundongos , Células Fotorreceptoras , Reação em Cadeia da Polimerase , Retina/efeitos dos fármacos , Retina/fisiopatologia , Proteínas de Ligação ao Retinol , Baço/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Uveíte/imunologia , Uveíte/fisiopatologiaRESUMO
Adoptive immunotherapy for solid tumors relies on infusing large numbers of T cells to mediate successful antitumor responses in patients. While long-term rapid-expansion protocols (REPs) produce sufficient numbers of CD8+ T cells for treatment, they also cause decline in the cell's therapeutic fitness. In contrast, we discovered that IL-17-producing CD4+ T cells (Th17 cells) do not require REPs to expand 5,000-fold over 3 weeks. Also, unlike Th1 cells, Th17 cells do not exhibit hallmarks of senescence or apoptosis, retaining robust antitumor efficacy in vivo. Three-week-expanded Th17 cells eliminated melanoma as effectively as Th17 cells expanded for 1 week when infused in equal numbers into mice. However, treating mice with large recalcitrant tumors required the infusion of all cells generated after 2 or 3 weeks of expansion, while the cell yield obtained after 1-week expansion was insufficient. Long-term-expanded Th17 cells also protected mice from tumor rechallenge including lung metastasis. Importantly, 2-week-expanded human chimeric antigen receptor-positive (CAR+) Th17 cells also retained their ability to regress human mesothelioma, while CAR+ Th1 cells did not. Our results indicate that tumor-reactive Th17 cells are an effective cell therapy for cancer, remaining uncompromised when expanded for a long duration owing to their resistance to senescence.
Assuntos
Senescência Celular , Imunoterapia Adotiva , Neoplasias Pulmonares/imunologia , Melanoma Experimental/imunologia , Mesotelioma/imunologia , Células Th17/citologia , Células Th17/imunologia , Animais , Linhagem Celular Tumoral , Humanos , Memória Imunológica , Neoplasias Pulmonares/terapia , Melanoma Experimental/terapia , Mesotelioma/terapia , Mesotelioma Maligno , Camundongos , Camundongos Endogâmicos C57BLRESUMO
A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase III. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by approximately 60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for 'repression of gene function' studies in vertebrate model systems.
Assuntos
Inativação Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Interferência de RNA , RNA Mensageiro/metabolismo , Xenopus laevis/genética , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Linhagem Celular , DNA , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Regiões Promotoras Genéticas , Retina/metabolismo , TransfecçãoRESUMO
The protein RPE65 is essential for the generation of the native chromophore, 11-cis retinal, of visual pigments. However, the Rpe65 knockout (Rpe65-/-) mouse shows a minimal visual response due to the presence of a pigment, isorhodopsin, formed with 9-cis retinal. Isorhodopsin accumulates linearly with prolonged dark-rearing of the animals. The majority of Rpe65-/- mice have an agouti coat color. A tan coat color subset of Rpe65-/- mice was found to have an enhanced visual response as measured by electroretinograms. The enhanced response was found to be due to increased levels of 9-cis retinal and isorhodopsin pigment levels. Animals of both coat colors reared in cyclic light have minimal levels of regenerated pigment and show photoreceptor degeneration. On dark-rearing, pigment accumulates and photoreceptor degeneration is decreased. In the tan Rpe65-/- mice, the level of photoreceptor degeneration is less than in the agouti animals, which have an increased pigment and decreased free opsin level. Therefore, photoreceptor damage correlates with the amount of the apoprotein present, supporting findings that the activity from unregenerated opsin can lead to photoreceptor degeneration.
Assuntos
Proteínas de Transporte/genética , Proteínas do Olho/genética , Retina/fisiologia , Retinaldeído/biossíntese , Animais , Diterpenos , Cor de Cabelo/genética , Isomerismo , Camundongos , Camundongos Knockout , Células Fotorreceptoras/patologia , Pigmentação/genética , Pigmentação/fisiologia , Retina/efeitos dos fármacos , Rodopsina/fisiologia , cis-trans-IsomerasesRESUMO
PURPOSE: RPE65 is essential for the generation of 11-cis retinal. Rod photoreceptors in the RPE65-knockout (Rpe65(-/-)) mouse are known to degenerate slowly with age. This study was designed to examine cone photoreceptors and the expression of cone-specific genes in the Rpe65(-/-) mouse. METHODS: Gene expression changes were identified by microarray and confirmed by real-time RT-PCR. Cone photoreceptors were stained by peanut agglutinin (PNA) lectin in the flatmounted retina. The 9- or 11-cis retinal was supplied by intraperitoneal injections. RESULTS: The short-wavelength (SWL) cone opsin mRNA was markedly decreased at 2 weeks of age, whereas the decrease in the middle-wavelength (MWL) cone opsin mRNA occurred relatively later in age. In contrast, the rhodopsin mRNA level did not show any significant change at all the ages analyzed. Consistent with the cone opsin changes, the cone transducin alpha-subunit mRNA decreased at both 4 and 8 weeks of age, whereas again the rod transducin alpha-subunit did not show any significant change. Rpe65(-/-) mice showed significant cone loss in both the central and ventral retina between 2 and 3 weeks of age. Administration of 9- or 11-cis retinal to Rpe65(-/-) mice 2 weeks of age increased cone density by twofold in these areas. CONCLUSIONS: In the Rpe65(-/-) mouse, the expression of cone-specific genes is downregulated and is accompanied by cone degeneration at early ages. Early administration of 9- or 11-cis retinal can partially prevent cone loss, suggesting that the absence of 11-cis chromophore may be responsible for the early cone degeneration.
Assuntos
Expressão Gênica , Proteínas/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/metabolismo , Opsinas de Bastonetes/genética , Animais , Proteínas de Transporte , Contagem de Células , Diterpenos , Regulação para Baixo , Proteínas do Olho , Subunidades alfa de Proteínas de Ligação ao GTP , Perfilação da Expressão Gênica , Proteínas Heterotriméricas de Ligação ao GTP/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Retinaldeído/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodopsina/genética , Transducina/genética , cis-trans-IsomerasesRESUMO
PURPOSE: In retinal degenerative diseases, rod photoreceptors typically deteriorate more rapidly than cone photoreceptors. In the Rpe65(-/-) mouse, a model for Leber's congenital amaurosis, cones degenerate much more rapidly than rods. In this model, the retinoid processing pathway in the retinal pigment epithelium is disrupted, and 11-cis retinal is not generated. This study was designed to investigate the feasibility of restoring functional cones with exogenous 11-cis retinal. METHODS: Rpe65(-/-)::Rho(-/-) mice were used to remove any interference of rods and compared with wild-type (wt) mice. Pups were injected intraperitoneally with 11-cis retinal, starting at postnatal day (P)10, and were maintained in complete darkness. At P25, cone function was assessed with photopic single-flash and flicker ERGs. Cone survival was determined immunohistochemically with cone-specific antibodies, and cone opsin levels were obtained by quantitative RT-PCR. RESULTS: At P25, cone density and transcript levels of cone opsins were drastically reduced, but a minute cone electroretinogram was detected, indicating that the cones were functional. Confocal microscopy revealed that the cone opsins were mislocalized, suggesting that their transport to the outer segments was impaired. Intraperitoneal administrations of 11-cis retinal before P25 led to increased transport of cone opsins to the outer segments and preserved cones anatomically and functionally. CONCLUSIONS: The results suggest that the ligand is required during cone opsin synthesis for successful opsin trafficking and that without 11-cis retinal, cones may degenerate because of opsin mislocalization. These results may have important consequences for the treatment of cone dystrophies.
Assuntos
Proteínas do Olho/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/metabolismo , Retinaldeído/farmacologia , Opsinas de Bastonetes/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte , Sobrevivência Celular , Eletrorretinografia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Estimulação Luminosa , RNA Mensageiro/metabolismo , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Degeneração Retiniana/fisiopatologia , Degeneração Retiniana/prevenção & controle , Retinaldeído/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodopsina/genética , Rodopsina/metabolismo , Opsinas de Bastonetes/genética , cis-trans-IsomerasesRESUMO
A novel conjugate of mitomycin C (MMC) and triamcinolone acetonide (TA) was synthesized using glutaric acid as a linker molecule. To determine the rate of hydrolysis, the conjugate was dissolved in aqueous solution and the rate of appearance of free MMC and TA was determined by high-performance liquid chromatography analysis. Antiproliferative activity of the MMC-TA conjugate and parent compounds was assessed using an NIH 3T3 fibroblast cell line. Cell growth was quantified using the MTT assay. Kinetic analysis of the hydrolysis rate demonstrated that the conjugate had a half-life of 23.6 h in aqueous solutions. The antiproliferative activities of the MMC-TA conjugate and MMC were both concentration dependent, with similar IC(50) values of 2.4 and 1.7 microM, respectively. However, individual responses at concentrations above 3 microM showed that the conjugate was less active than MMC alone. TA alone showed only limited inhibition of cell growth. Studies evaluating intravitreal injection of the conjugate demonstrate that this agent produced no measurable toxicity. Our data provide evidence that the MMC-TA conjugate could be used as a slow-release drug delivery system. This could in turn be used to modulate a posttreatment wound healing process or to treat various proliferative diseases.