RESUMEN
γ2 adaptin is homologous to γ1, but is only expressed in vertebrates while γ1 is found in all eukaryotes. We know little about γ2 functions and their relation to γ1. γ1 is an adaptin of the heterotetrameric AP-1 complexes, which sort proteins in and do form clathrin-coated transport vesicles and they also regulate maturation of early endosomes. γ1 knockout mice develop only to blastocysts and thus γ2 does not compensate γ1-deficiency in development. γ2 has not been classified as a clathrin-coated vesicle adaptor protein in proteome analyses and functions for monomeric γ2 in endosomal protein sorting have been proposed, but adaptin interaction studies suggested formation of heterotetrameric AP-1/γ2 complexes. We detected γ2 at the trans-Golgi network, on peripheral vesicles and identified γ2 clathrin-coated vesicles in mice. Ubiquitous σ1A and tissue-specific σ1B adaptins bind γ2 and γ1. σ1B knockout in mice does not effect γ1/σ1A AP-1 levels, but γ2/σ1A AP-1 levels are increased in brain and adipocytes. Also γ2 is essential in development. In zebrafish AP-1/γ2 and AP-1/γ1 fulfill different, essential functions in brain and the vascular system.
Asunto(s)
Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Encéfalo/metabolismo , Clatrina/metabolismo , Vesículas Transportadoras/metabolismo , Red trans-Golgi/metabolismo , Subunidades gamma de Complejo de Proteína Adaptadora/química , Subunidades gamma de Complejo de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/química , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Adipocitos/citología , Adipocitos/metabolismo , Animales , Vasos Sanguíneos/crecimiento & desarrollo , Vasos Sanguíneos/metabolismo , Encéfalo/crecimiento & desarrollo , Línea Celular , Clatrina/genética , Embrión de Mamíferos , Embrión no Mamífero , Endosomas/metabolismo , Endosomas/ultraestructura , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Modelos Moleculares , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , Transporte de Proteínas/genética , Transducción de Señal , Vesículas Transportadoras/ultraestructura , Pez Cebra , Red trans-Golgi/ultraestructuraRESUMEN
Here, we describe altered sorting of sortilin in adipocytes deficient for the σ1B-containing AP-1 complex, leading to the inhibition of adipogenesis. The AP-1 complex mediates protein sorting between the trans-Golgi network and endosomes. Vertebrates express three AP1 σ1 subunit isoforms - σ1A, σ1B and σ1C (also known as AP1S1, AP1S2 and AP1S3, respectively). σ1B-deficient mice display impaired recycling of synaptic vesicles and lipodystrophy. Here, we show that sortilin is overexpressed in adipose tissue from σ1B(-/-) mice, and that its overexpression in wild-type cells is sufficient to suppress adipogenesis. σ1B-specific binding of sortilin requires the sortilin DxxD-x12-DSxxxL motif. σ1B deficiency does not lead to a block of sortilin transport out of a specific organelle, but the fraction that reaches lysosomes is reduced. Sortilin binds to the receptor DLK1, an inhibitor of adipocyte differentiation, and the overexpression of sortilin prevents DLK1 downregulation, leading to enhanced inhibition of adipogenesis. DLK1 and sortilin expression are not increased in the brain tissue of σ1B(-/-) mice, although this is the tissue with the highest expression of σ1B and sortilin. Thus, adipose-tissue-specific and σ1B-dependent routes for the transport of sortilin exist and are involved in the regulation of adipogenesis and adipose-tissue mass.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Adipocitos/metabolismo , Adipogénesis , Tejido Adiposo/metabolismo , Complejo 1 de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Adipocitos/citología , Tejido Adiposo/citología , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de ProteínasRESUMEN
Synapsins are abundant synaptic vesicle (SV)-associated proteins thought to mediate synaptic vesicle mobility and clustering at most synapses. We used synapsin triple knock-out (TKO) mice to examine the morphological and functional consequences of deleting all synapsin isoforms at the calyx of Held, a giant glutamatergic synapse located in the auditory brain stem. Quantitative three-dimensional (3D) immunohistochemistry of entire calyces showed lower amounts of the synaptic vesicle protein vGluT1 while the level of the active zone marker bassoon was unchanged in TKO terminals. Examination of brain lysates by ELISA revealed a strong reduction in abundance of several synaptic vesicle proteins, while proteins of the active zone cytomatrix or postsynaptic density were unaffected. Serial section scanning electron microscopy of large 3D-reconstructed segments confirmed a decrease in the number of SVs to approximately 50% in TKO calyces. Short-term depression tested at stimulus frequencies ranging from 10 to 300 Hz was accelerated only at frequencies above 100 Hz and the time course of recovery from depression was slowed in calyces lacking synapsins. These results reveal that in wild-type synapses, the synapsin-dependent reserve pool contributes to the replenishment of the readily releasable pool (RRP), although accounting only for a small fraction of the SVs that enter the RRP. In conclusion, our results suggest that synapsins may be required for normal synaptic vesicle biogenesis, trafficking and immobilization of synaptic vesicles, yet they are not essential for sustained high-frequency synaptic transmission at the calyx terminal.
Asunto(s)
Sinapsinas/genética , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Animales , Tronco Encefálico/metabolismo , Exocitosis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Isoformas de Proteínas/genética , Sinapsis/metabolismo , Sinapsis/fisiología , Potenciales Sinápticos , Transmisión Sináptica/genética , Proteína 1 de Transporte Vesicular de Glutamato/genética , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
N-terminally truncated Aß peptides starting with pyroglutamate (AßpE3) represent a major fraction of all Aß peptides in the brain of Alzheimer disease (AD) patients. AßpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aß. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AßpE3 and studied the potential involvement of oligomeric AßpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aß plaque load and AßpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AßpE3 oligomers.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Ácido Pirrolidona Carboxílico/química , Amiloide/química , Animales , Conducta Animal , Encéfalo/metabolismo , Línea Celular Tumoral , Cromatografía/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inmunoquímica/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Peso Molecular , Estructura Terciaria de Proteína , TransgenesRESUMEN
Enzyme-linked immunosorbent assays (ELISAs) are applied for the quantification of a vast diversity of small molecules. However, ELISAs require that the antigen is present in a soluble form in the sample. Accordingly, the few ELISAs described so far targeting insoluble proteins such as integral membrane and scaffold proteins have been restricted by limited extraction efficiencies and the need to establish an individual solubilization protocol for each protein. Here we describe a sandwich ELISA that allows the quantification of a diverse array of synaptic membrane and scaffold proteins such as munc13-1, gephyrin, NMDA R1 (N-methyl-d-aspartate receptor subunit 1), synaptic vesicle membrane proteins, and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The assay is based on initial solubilization by the denaturing detergent sodium dodecyl sulfate (SDS), followed by partial SDS removal using the detergent Triton X-100, which restores antigenicity while keeping the proteins in solution. Using recombinant standard proteins, we determined assay sensitivities of 78ng/ml to 77pg/ml (or 74-0.1fmol). Calibration of the assay using both immunoblotting and mass spectroscopy revealed that in some cases correction factors need to be included for absolute quantification. The assay is versatile, allows parallel processing and automation, and should be applicable to a wide range of hitherto inaccessible proteins.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas de la Membrana/análisis , Membranas Sinápticas/química , Animales , Calibración , Detergentes , Inmunoprecipitación , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Ratones , Ratas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , SolubilidadRESUMEN
Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1-sigma1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1-sigma1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three sigma1 subunit isoforms: A, B and C. The expressions of sigma1A and sigma1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from sigma1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The sigma1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation.