RESUMEN
Early endosome antigen 1 (EEA1), a peripheral membrane protein associated with the cytoplasmic face of early endosomes, controls vesicle fusion during endocytosis, as extensively studied in non-neuronal cells. In neurons, early endosomes are involved in recycling of synaptic vesicles and neurotransmitter receptors. Since certain patients bearing autoantibodies that target EEA1 develop neurological disease, we studied the subcellular distribution of EEA1 in neurons and the effect on neurotransmission of purified immunoglobulins from the serum of a patient bearing EEA1 autoantibodies. EEA1 was localized in the soma and in the postsynaptic nerve terminals. Electrophysiological recordings in hippocampal slices including purified EEA1 antibodies in the patch pipette solution, revealed a run-up of AMPA, N-methyl-D-aspartate and kainate receptor-mediated excitatory post-synaptic currents recorded from CA3 pyramidal neurons, which was absent in the recordings obtained in the presence of control human immunoglobulin G. Inclusion of human EEA1 antibodies had no effect on inhibitory post-synaptic responses. Recordings in the presence of a dominant-negative C-terminal EEA1 deletion mutant produced a similar effect as observed with human anti-EEA1 antibodies. This specific effect on the excitatory synaptic transmission may be due to the impairment of internalization of specific glutamate receptors and their subsequent accumulation in the synapse. These results may account for the neurological deficits observed in some patients developing EEA1 autoantibodies.
Asunto(s)
Autoanticuerpos/inmunología , Potenciales Postsinápticos Excitadores/inmunología , Hipocampo/inmunología , Proteínas de la Membrana/inmunología , Células Piramidales/inmunología , Transmisión Sináptica/inmunología , Proteínas de Transporte Vesicular/inmunología , Animales , Autoanticuerpos/farmacología , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Enfermedades Autoinmunes del Sistema Nervioso/fisiopatología , Células COS , Chlorocebus aethiops , Endocitosis/genética , Endocitosis/inmunología , Femenino , Hipocampo/metabolismo , Hipocampo/fisiopatología , Humanos , Inmunohistoquímica , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Mutación/genética , Mutación/inmunología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Células Piramidales/metabolismo , Agregación de Receptores/genética , Agregación de Receptores/inmunología , Receptores de Glutamato/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Autoantibodies to EEA1 have been described in patients with neurological diseases, subacute cutaneous lupus and a variety of other conditions, including a patient with Wegener's granulomatosis (WG). EEA1 is a hydrophilic peripheral membrane protein transiently associated with the cytoplasmic face of early endosomes. Antibodies to EEA1 produce a staining pattern that resembles the C-ANCA pattern produced by anti-proteinase 3 (PR3) antibodies in WG sera. Co-localization studies show incomplete overlap of the staining produced by anti-EEA1 with anti-PR3. We showed that 0/40 unselected sera, from a cohort of WG patients and antibodies to PR3, reacted with EEA1. In addition, 1/15 sera that have a C-ANCA staining pattern but do not react with PR3 in an ELISA, immunoprecipitated the recombinant EEA1 protein. We conclude that although antibodies to EEA1 produce a staining pattern that resembles anti-PR3 and C-ANCA, antibodies to EEA1 in WG are rare. However, some C-ANCA+ sera that do not react with PR3 may contain EEA1 autoantibodies.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Granulomatosis con Poliangitis/inmunología , Proteínas de la Membrana/inmunología , Animales , Autoanticuerpos/sangre , Electroforesis en Gel de Poliacrilamida/métodos , Técnica del Anticuerpo Fluorescente Indirecta , Granulomatosis con Poliangitis/sangre , Humanos , Immunoblotting/métodos , Mieloblastina , Neutrófilos/inmunología , Pruebas de Precipitina/métodos , Conejos , Serina Endopeptidasas/inmunología , Dodecil Sulfato de Sodio , Coloración y Etiquetado , Células Tumorales Cultivadas , Proteínas de Transporte VesicularRESUMEN
BACKGROUND: We have identified 36 human sera sent for autoantibody analyses that produce a unique vesicular staining pattern of the cytoplasm of tissue culture cells. The purpose of this study was to identify the autoantigens that are recognized by the sera that produce this staining pattern and determine if the patients have common clinical features. METHODS: A serum from one of the patients (MS) with rapidly progressive demyelinating polyneuropathy was used to isolate a approximately 4.5 kb cDNA insert from a HeLa expression library. The purified cDNA (MS-5.1) was characterized by a poly A tail and an open reading frame that encoded 1329 amino acids. The derived amino acid sequence was found to be 99% identical to a 180 kd peripheral endosomal protein named early endosome antigen (EEA1). RESULTS: Antibodies from rabbits immunized with the recombinant protein and the prototype human serum produced an identical distinctive speckled cytoplasmic staining pattern. These sera also precipitated the in vitro translated recombinant protein and reacted with the isolated recombinant protein in a Western immunoblot. Of the 36 sera that produced an identical staining pattern as the prototype and immune rabbit sera, 8 (22%) had IgG antibodies that recognized the recombinant EEA1 protein when tested by immunoblotting and immunoprecipitation assays. Of the 8 patients with anti-EEA1 antibodies 4 were females, 4 were males, and the mean age was 69 years (range 48 to 86 years). CONCLUSIONS: Diagnoses included: polyneuropathy, lower motor neuron disease, pigmented retinitis, seronegative polyarthritis, interstitial pulmonary fibrosis, Raynaud's phenomenon, Wegener's granulomatosis, and proteinuria. Three of the eight patients with EEA1 autoantibodies died within 1 year after EEA1 antibodies were identified.