RESUMO
Lysosomal diseases are a class of genetic disorders predominantly caused by loss of lysosomal hydrolases, leading to lysosomal and cellular dysfunction. Enzyme replacement therapy (ERT), where recombinant enzyme is given intravenously, internalized by cells, and trafficked to the lysosome, has been applied to treat several lysosomal diseases. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of the affected cells that require the enzyme. We present here targeted ERT, an approach that utilizes antibody-enzyme fusion proteins to target the enzyme to specific cell types. The antibody moiety recognizes transmembrane proteins involved in lysosomal trafficking and that are also preferentially expressed in those cells most affected in disease. Using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice both as a protein replacement therapeutic and as a gene therapy.
Assuntos
Doença de Depósito de Glicogênio Tipo II , Doenças por Armazenamento dos Lisossomos , Animais , Terapia de Reposição de Enzimas , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo II/genética , Hidrolases/metabolismo , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/genética , Lisossomos/metabolismo , Camundongos , Distribuição Tecidual , alfa-Glucosidases/genéticaRESUMO
RATIONALE: Neointimal hyperplasia characterized by abnormal accumulation of vascular smooth muscle cells (SMCs) is a hallmark of occlusive disorders such as atherosclerosis, postangioplasty restenosis, vein graft stenosis, and allograft vasculopathy. Cyclic nucleotides are vital in SMC proliferation and migration, which are regulated by cyclic nucleotide phosphodiesterases (PDEs). OBJECTIVE: Our goal is to understand the regulation and function of PDEs in SMC pathogenesis of vascular diseases. METHODS AND RESULTS: We performed screening for genes differentially expressed in normal contractile versus proliferating synthetic SMCs. We observed that PDE1C expression was low in contractile SMCs but drastically elevated in synthetic SMCs in vitro and in various mouse vascular injury models in vivo. In addition, PDE1C was highly induced in neointimal SMCs of human coronary arteries. More importantly, injury-induced neointimal formation was significantly attenuated by PDE1C deficiency or PDE1 inhibition in vivo. PDE1 inhibition suppressed vascular remodeling of human saphenous vein explants ex vivo. In cultured SMCs, PDE1C deficiency or PDE1 inhibition attenuated SMC proliferation and migration. Mechanistic studies revealed that PDE1C plays a critical role in regulating the stability of growth factor receptors, such as PDGF receptor ß (PDGFRß) known to be important in pathological vascular remodeling. PDE1C interacts with low-density lipoprotein receptor-related protein-1 and PDGFRß, thus regulating PDGFRß endocytosis and lysosome-dependent degradation in an low-density lipoprotein receptor-related protein-1-dependent manner. A transmembrane adenylyl cyclase cAMP-dependent protein kinase cascade modulated by PDE1C is critical in regulating PDGFRß degradation. CONCLUSIONS: These findings demonstrated that PDE1C is an important regulator of SMC proliferation, migration, and neointimal hyperplasia, in part through modulating endosome/lysosome-dependent PDGFRß protein degradation via low-density lipoprotein receptor-related protein-1.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/enzimologia , Neointima/enzimologia , Animais , Lesões das Artérias Carótidas/enzimologia , Lesões das Artérias Carótidas/patologia , Divisão Celular , Movimento Celular , Células Cultivadas , AMP Cíclico/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/antagonistas & inibidores , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/deficiência , Endocitose/fisiologia , Indução Enzimática , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Lisossomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Miócitos de Músculo Liso/citologia , Neointima/fisiopatologia , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Proteólise , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Feedback inhibition of adenylyl cyclase III (ACIII) via Ca(2+)-induced phosphorylation has long been hypothesized to contribute to response termination and adaptation of olfactory sensory neurons (OSNs). To directly determine the functional significance of this feedback mechanism for olfaction in vivo, we genetically mutated serine(1076) of ACIII, the only residue responsible for Ca(2+)-induced phosphorylation and inhibition of ACIII (Wei et al., 1996, 1998), to alanine in mice. Immunohistochemistry and Western blot analysis showed that the mutation affects neither the cilial localization nor the expression level of ACIII in OSNs. Electroolfactogram analysis showed no differences in the responses between wild-type and mutant mice to single-pulse odorant stimulations or in several stimulation paradigms for adaptation. These results suggest that phosphorylation of ACIII on serine(1076) plays a far less important role in olfactory response attenuation than previously thought.
Assuntos
Adenilil Ciclases/metabolismo , Nervo Olfatório/enzimologia , Serina/genética , Olfato/genética , Adenilil Ciclases/genética , Sequência de Aminoácidos , Animais , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Nervo Olfatório/metabolismo , Fosforilação/genéticaRESUMO
Mucopolysaccharidosis VI (MPS VI) is a rare lysosomal disease arising from impaired function of the enzyme arylsulfatase B (ARSB). This impairment causes aberrant accumulation of dermatan sulfate, a glycosaminoglycan (GAG) abundant in cartilage. While clinical severity varies along with age at first symptom manifestation, MPS VI usually presents early and strongly affects the skeleton. Current enzyme replacement therapy (ERT) does not provide effective treatment for the skeletal manifestations of MPS VI. This lack of efficacy may be due to an inability of ERT to reach affected cells or to the irreversibility of the disease. To address the question of reversibility of skeletal phenotypes, we generated a conditional by inversion (COIN) mouse model of MPS VI, ArsbCOIN/COIN, wherein Arsb is initially null and can be restored to WT using Cre. We restored Arsb at different times during postnatal development, using a tamoxifen-dependent global Cre driver. By restoring Arsb at P7, P21, and P56-P70, we determined that skeletal phenotypes can be fully rescued if Arsb restoration occurs at P7, while only achieving partial rescue at P21 and no significant rescue at P56-P70. This work has highlighted the importance of early intervention in patients with MPS VI to maximize therapeutic impact.
Assuntos
Mucopolissacaridose VI , N-Acetilgalactosamina-4-Sulfatase , Camundongos , Animais , Humanos , Mucopolissacaridose VI/tratamento farmacológico , Mucopolissacaridose VI/diagnóstico , N-Acetilgalactosamina-4-Sulfatase/genética , N-Acetilgalactosamina-4-Sulfatase/uso terapêutico , Fenótipo , Glicosaminoglicanos , EsqueletoRESUMO
For vertebrate olfactory signal transduction, a calcium-activated chloride conductance serves as a major amplification step. However, the molecular identity of the olfactory calcium-activated chloride channel (CaCC) is unknown. Here we report a proteomic screen for cilial membrane proteins of mouse olfactory sensory neurons (OSNs) that identified all the known olfactory transduction components as well as Anoctamin 2 (ANO2). Ano2 transcripts were expressed specifically in OSNs in the olfactory epithelium, and ANO2::EGFP fusion protein localized to the OSN cilia when expressed in vivo using an adenoviral vector. Patch-clamp analysis revealed that ANO2, when expressed in HEK-293 cells, forms a CaCC and exhibits channel properties closely resembling the native olfactory CaCC. Considering these findings together, we propose that ANO2 constitutes the olfactory calcium-activated chloride channel.
Assuntos
Canais de Cloreto/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Olfato/fisiologia , Animais , Anoctaminas , Linhagem Celular , Cílios/metabolismo , Humanos , Camundongos , Neurônios Receptores Olfatórios/metabolismo , Técnicas de Patch-Clamp , ProteômicaRESUMO
The protein phosphatase 2A (PP2A) regulatory subunit Tap42 is essential for target of rapamycin (TOR)-mediated signaling in yeast, but its role in higher eukaryotes has not been established. Here we show that Tap42 does not contribute significantly to TOR signaling in Drosophila, as disruption of the Tap42 gene does not cause defects in cell growth, metabolism, or S6-kinase activity characteristic of TOR inactivation. In addition, Tap42 is not required for increased cell growth in response to activation of TOR signaling. Instead, we find that Tap42 mutations cause disorganization of spindle microtubules in larval neuroblasts, leading to a preanaphase mitotic arrest in these cells. Loss of Tap42 ultimately results in increased JNK signaling, caspase activation, and cell death. These phenotypes are associated with increased accumulation and nuclear localization of PP2A in Tap42 mutant cells. Our results demonstrate that the role of Tap42 in TOR signaling has not been conserved in higher eukaryotes, indicating fundamental differences in the mechanisms of TOR signaling between yeast and higher eukaryotes.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Ciclo Celular , Morte Celular , Divisão Celular , Núcleo Celular/metabolismo , Proliferação de Células , Separação Celular , Sobrevivência Celular , Sequência Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Ativação Enzimática , Citometria de Fluxo , Vetores Genéticos , Humanos , Microtúbulos/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases , Proteína Fosfatase 2 , Estrutura Terciária de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Especificidade da Espécie , Fuso Acromático , Serina-Treonina Quinases TOR , TransgenesRESUMO
Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses. Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.
Assuntos
Eletrofisiologia/métodos , Mucosa Olfatória/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Animais , Eletrodos , Eletrofisiologia/instrumentação , Camundongos , OdorantesRESUMO
In the nose, odorants are detected on the cilia of olfactory sensory neurons (OSNs), where a cAMP-mediated signaling pathway transforms odor stimulation into electrical responses. Phosphodiesterase (PDE) activity in OSN cilia has long been thought to account for rapid response termination by degrading odor-induced cAMP. Two PDEs with distinct cellular localization have been found in OSNs: PDE1C in the cilia and PDE4A throughout the cell but absent from the cilia. We disrupted both of these genes in mice and carried out electro-olfactogram analysis. Unexpectedly, eliminating PDE1C did not prolong response termination. Prolonged termination occurred only in mice that lacked both PDEs, suggesting that cAMP degradation by PDE1C in cilia is not a rate-limiting factor for response termination in wild-type mice. Pde1c(-/-) OSNs instead showed reduced sensitivity and attenuated adaptation to repeated stimulation, suggesting that PDE1C may be involved in regulating sensitivity and adaptation. Our observations provide new perspectives on the regulation of olfactory transduction.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Potenciais Evocados/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Tempo de Reação/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Animais , Cálcio/farmacologia , Calmodulina/farmacologia , Simulação por Computador , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/deficiência , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/deficiência , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Relação Dose-Resposta a Droga , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/genética , Camundongos , Camundongos Knockout , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/efeitos dos fármacos , Pentanóis/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/genéticaRESUMO
Ca2+/calmodulin-mediated negative feedback is a prototypical regulatory mechanism for Ca2+-permeable ion channels. In olfactory sensory neurons (OSNs), such regulation on the cyclic nucleotide-gated (CNG) channel is considered a major mechanism of OSN adaptation. To determine the role of Ca2+/calmodulin desensitization of the olfactory CNG channel, we introduced a mutation in the channel subunit CNGB1b in mice that rendered the channel resistant to fast desensitization by Ca2+/calmodulin. Contrary to expectations, mutant OSNs showed normal receptor current adaptation to repeated stimulation. Rather, they displayed slower response termination and, consequently, reduced ability to transmit olfactory information to the olfactory bulb. They also displayed reduced response decline during sustained odorant exposure. These results suggest that Ca2+/calmodulin-mediated CNG channel fast desensitization is less important in regulating the sensitivity to recurring stimulation than previously thought and instead functions primarily to terminate OSN responses.