RESUMO
Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible.
Assuntos
Dependovirus/genética , Edição de Genes/métodos , Células Fotorreceptoras de Vertebrados/fisiologia , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Animais , Sistemas CRISPR-Cas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/deficiência , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Reparo do DNA por Junção de Extremidades , Proteínas do Olho/genética , Terapia Genética/métodos , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Distrofias Retinianas/fisiopatologia , Reparo Gênico Alvo-Dirigido/métodos , Transducina/deficiência , Transducina/genética , Acuidade Visual/genética , Acuidade Visual/fisiologiaRESUMO
Purpose: The purpose of this study was to test the extent of light damage in different models of night blindness and apply these paradigms in testing the therapeutic efficacy of combination therapy by drugs acting on the Gi, Gs, and Gq protein-coupled receptors. Methods: Acute bright light exposure was used to test susceptibility to light damage in mice lacking the following crucial phototransduction proteins: rod transducin (GNAT1), cone transducin (GNAT2), visual arrestin 1 (ARR1), and rhodopsin kinase 1 (GRK1). Mice were intraperitoneally injected with either vehicle or drug combination consisting of metoprolol (ß1-receptor antagonist), bromocriptine (dopamine family-2 receptor agonist) and tamsulosin (α1-receptor antagonist) before bright light exposure. Light damage was primarily assessed with optical coherence tomography and inspection of cone population in retinal whole mounts. Retinal inflammation was assessed in a subset of experiments using autofluorescence imaging by scanning laser ophthalmoscopy and by postmortem inspection of microglia and astrocyte activity. Results: The Gnat1-/- mice showed slightly increased susceptibility to rod light damage, whereas the Gnat2-/- mice were very resistant. The Arr1-/- and Grk1-/- mice were sensitive for both rod and cone light damage and showed robust retinal inflammation 7 days after bright light exposure. Pretreatment with metoprolol + bromocriptine + tamsulosin rescued the retina in all genetic backgrounds, starting at doses of 0.2 mg/kg metoprolol, 0.02 mg/kg bromocriptine, and 0.01 mg/kg tamsulosin in the Gnat1-/- mice. The therapeutic drug doses increased in parallel with light-damage severity. Conclusions: Our results suggest that congenital stationary night blindness and Oguchi disease patients can be at an elevated risk of the toxic effects of bright light. Furthermore, systems pharmacology drug regimens that stimulate Gi signaling and attenuate Gs and Gq signaling present a promising disease-modifying therapy for photoreceptor degenerative diseases.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 1/uso terapêutico , Antagonistas de Receptores Adrenérgicos beta 1/uso terapêutico , Bromocriptina/farmacologia , Luz/efeitos adversos , Metoprolol/farmacologia , Cegueira Noturna/tratamento farmacológico , Tansulosina/farmacologia , Animais , Arrestinas/deficiência , Modelos Animais de Doenças , Receptor Quinase 1 Acoplada a Proteína G/deficiência , Camundongos , Transducina/deficiência , Estados Unidos , United States Food and Drug AdministrationRESUMO
Ginsenosides can be classified on the basis of the skeleton of their aglycones. Here, we hypothesized that the sugar moieties attached to the dammarane backbone enable binding of the ginsenosides to the sweet taste receptor, eliciting glucagon-like peptide-1 (GLP-1) secretion in the enteroendocrine L cells. Using the human enteroendocrine NCI-H716 cells, we demonstrated that 15 ginsenosides stimulate GLP-1 secretion according to the position of their sugar moieties. Through a pharmacological approach and RNA interference technique to inhibit the cellular signal cascade and using the Gαgust(-/-) mice, we elucidated that GLP-1 secreting effect of Rg3 mediated by the sweet taste receptor mediated the signaling pathway. Rg3, a ginsenoside metabolite that transformed the structure through a steaming process, showed the strongest GLP-1 secreting effects in NCI-H716 cells and also showed an anti-hyperglycemic effect on a type 2 diabetic mouse model through increased plasma GLP-1 and plasma insulin levels during an oral glucose tolerance test. Our study reveals a novel mechanism where the sugar moieties of ginsenosides Rg3 stimulates GLP-1 secretion in enteroendocrine L cells through a sweet taste receptor-mediated signal transduction pathway and thus has an anti-hyperglycemic effect on the type 2 diabetic mouse model.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Células Enteroendócrinas/efeitos dos fármacos , Ginsenosídeos/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hiperglicemia/prevenção & controle , Animais , Linhagem Celular Tumoral , Células Enteroendócrinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/sangue , Teste de Tolerância a Glucose , Humanos , Hiperglicemia/complicações , Immunoblotting , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transducina/deficiência , Transducina/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer fatalities in Western societies, characterized by high metastatic potential and resistance to chemotherapy. Critical molecular mechanisms of these phenotypical features still remain unknown, thus hampering the development of effective prognostic and therapeutic measures in PDAC. Here, we show that transcriptional co-factor Transducin beta-like (TBL) 1 was over-expressed in both human and murine PDAC. Inactivation of TBL1 in human and mouse pancreatic cancer cells reduced cellular proliferation and invasiveness, correlating with diminished glucose uptake, glycolytic flux, and oncogenic PI3 kinase signaling which in turn could rescue TBL1 deficiency-dependent phenotypes. TBL1 deficiency both prevented and reversed pancreatic tumor growth, mediated transcriptional PI3 kinase inhibition, and increased chemosensitivity of PDAC cells in vivo. As TBL1 mRNA levels were also found to correlate with PI3 kinase levels and overall survival in a cohort of human PDAC patients, TBL1 was identified as a checkpoint in the malignant behavior of pancreatic cancer and its expression may serve as a novel molecular target in the treatment of human PDAC.
Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Transducina/metabolismo , Animais , Perfilação da Expressão Gênica , Humanos , Camundongos , Análise de Sobrevida , Transducina/deficiênciaRESUMO
Visual input in the critical period is an important determinant of the functions of the visual system, affecting for example the formation of the ocular dominance column in the visual cortex. The final map of columnar organization is usually determined by plastic changes in the critical period, but organization is distorted without adequate visual input. Here, we examined whether formation of the OFF-pathway dominance of P2X(2)-purinoceptor signaling in the mouse retina is the result of visual experience. The P2X(2)-purinoceptor signaling pathway developed during the critical period. However, visual experience in this period produced no plastic change in the formation of the OFF-pathway dominance of P2X(2)-purinoceptor signaling. Our findings suggest that the OFF-pathway dominance of P2X(2)-signaling in the mouse retina is intrinsically programmed.
Assuntos
Células Amácrinas/fisiologia , Inibição Neural/fisiologia , Receptores Purinérgicos P2/metabolismo , Retina/citologia , Transdução de Sinais/fisiologia , Córtex Visual/fisiologia , Trifosfato de Adenosina/farmacologia , Células Amácrinas/efeitos dos fármacos , Animais , Colina O-Acetiltransferase/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/deficiência , Técnicas In Vitro , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Inibição Neural/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2 , Opsinas de Bastonetes/deficiência , Transdução de Sinais/genética , Transducina/deficiência , Vias Visuais/fisiologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
We have used wild-type mice and mice possessing defects in specific retinal circuits in order to more clearly define functional circuits of the inner retina. The retina of the nob mouse lacks communication between photoreceptors and depolarizing bipolar cells (DBCs). Thus, all light driven activity in the nob mouse is mediated via remaining hyperpolarizing bipolar cell (HBC) circuits. Transducin null (Tr alpha-/-) mice lack rod photoreceptor activity and thus remaining retinal circuits are solely generated via cone photoreceptor activity. Activation in inner retinal circuits in each of these mice was identified by monitoring light-induced expression of an immediate early gene, c-fos. The number of cells expressing c-fos in the inner retina was dependent upon stimulus intensity and was altered in a systematic fashion in mice with known retinal mutations. To determine whether c-fos is activated via circuits other than photoreceptors in the outer retina, we examined c-fos expression in tulp1-/- mice that lack photoreceptors in the outer retina; these mice showed virtually no c-fos activity following light exposure. Double-labeling immunohistochemical studies were carried out to more clearly define the population of c-fos expressing amacrine cells. Our results indicate that c-fos may be used to map functional circuits in the retina.