RESUMEN

Synapse loss and Tau pathology are hallmarks of Alzheimer's disease (AD) and other tauopathies, but how Tau pathology causes synapse loss is unclear. We used unbiased proteomic analysis of postsynaptic densities (PSDs) in Tau-P301S transgenic mice to identify Tau-dependent alterations in synapses prior to overt neurodegeneration. Multiple proteins and pathways were altered in Tau-P301S PSDs, including depletion of a set of GTPase-regulatory proteins that leads to actin cytoskeletal defects and loss of dendritic spines. Furthermore, we found striking accumulation of complement C1q in the PSDs of Tau-P301S mice and AD patients. At synapses, C1q decorated perisynaptic membranes, accumulated in correlation with phospho-Tau, and was associated with augmented microglial engulfment of synapses and decline of synapse density. A C1q-blocking antibody inhibited microglial synapse removal in cultured neurons and in Tau-P301S mice, rescuing synapse density. Thus, inhibiting complement-mediated synapse removal by microglia could be a potential therapeutic target for Tau-associated neurodegeneration.

Asunto(s)

Anticuerpos/uso terapéutico , Complemento C1q/inmunología , Sinapsis/metabolismo , Tauopatías/tratamiento farmacológico , Tauopatías/patología , Proteínas tau/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular , Células Cultivadas , Complemento C1q/metabolismo , Complemento C1q/ultraestructura , Embrión de Mamíferos , Femenino , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Densidad Postsináptica/metabolismo , Densidad Postsináptica/patología , Densidad Postsináptica/ultraestructura , Presenilina-2/genética , Presenilina-2/metabolismo , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Proteoma/metabolismo , Ratas , Sinapsis/efectos de los fármacos , Sinapsis/ultraestructura , Tauopatías/diagnóstico por imagen , Tauopatías/genética , Proteínas tau/genética

RESUMEN

C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K(D) = 0.34-2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response.

Asunto(s)

Membrana Celular/enzimología , Complemento C1q/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Streptococcus pneumoniae/enzimología , Apoptosis , Estructuras de la Membrana Celular/metabolismo , Activación de Complemento , Complemento C1q/química , Complemento C1q/ultraestructura , Gliceraldehído-3-Fosfato Deshidrogenasas/ultraestructura , Células HeLa , Humanos , Proteínas Inmovilizadas/metabolismo , Cinética , Ligandos , Mutación/genética , Plasminógeno/metabolismo , Unión Proteica , Transporte de Proteínas , Solubilidad , Soluciones , Resonancia por Plasmón de Superficie

RESUMEN

C1q nephropathy (C1qNP) is a peculiar form of glomerulonephritis characterized by mesangial immunoglobulin and complement deposits, predominantly C1q, with no evidence of systemic lupus erythematosus. We describe the incidence, manifestation, histopathologic findings, follow-up, treatment and outcome of C1qNP. Twelve C1qNP patients were identified among 131 children who had undergone renal biopsy, accounting for a 9.16% incidence of C1qNP. Light microscopy examination showed focal segmental glomerulosclerosis (FSGS) with or without diffuse mesangial proliferation (n=6), minimal change disease (MCD) (n=4) or focal glomerulonephritis (n=2). C1q deposits were found in all, while electron microscopy revealed visible deposits in nine cases. Eight children presented with nephrotic syndrome, while one had nephrotic proteinuria and renal insufficiency that progressed to end-stage renal failure. The remaining three patients presented with nonnephrotic proteinuria associated with microhematuria, hypertension or renal insufficiency. Only one nephrotic syndrome patient responded excellently to corticosteroids, while four became corticosteroid dependent, and three were corticosteroid resistant, showing a very poor response to other immunosuppressive therapy as well. Patients with non-nephrotic proteinuria demonstrated fixed laboratory findings. Most C1qNP patients had FSGS or MCD, the majority of them presenting with corticosteroid-dependent or corticosteroid-resistant nephrotic syndrome. The latter showed a very poor response to any immunosuppressive therapy and high risk for progressive renal insufficiency.

Asunto(s)

Complemento C1q/metabolismo , Glomerulonefritis/diagnóstico , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Riñón/metabolismo , Riñón/patología , Adolescente , Corticoesteroides/uso terapéutico , Biopsia , Niño , Preescolar , Estudios de Cohortes , Complemento C1q/análisis , Complemento C1q/ultraestructura , Femenino , Técnica del Anticuerpo Fluorescente Directa , Estudios de Seguimiento , Mesangio Glomerular/metabolismo , Mesangio Glomerular/patología , Mesangio Glomerular/ultraestructura , Glomerulonefritis/tratamiento farmacológico , Glomerulonefritis/epidemiología , Glomerulonefritis/fisiopatología , Glomerulonefritis/cirugía , Humanos , Hipertensión/etiología , Inmunosupresores/uso terapéutico , Incidencia , Riñón/fisiopatología , Riñón/cirugía , Riñón/ultraestructura , Masculino , Síndrome Nefrótico/tratamiento farmacológico , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patología , Pronóstico , Proteinuria/tratamiento farmacológico , Proteinuria/metabolismo , Proteinuria/fisiopatología , Insuficiencia Renal/tratamiento farmacológico , Insuficiencia Renal/metabolismo , Insuficiencia Renal/patología , Estudios Retrospectivos , Prevención Secundaria , Eslovenia/epidemiología , Factores de Tiempo

Asunto(s)

Congelación , Microscopía de Fuerza Atómica , Actinas/ultraestructura , Animales , Complemento C1q/ultraestructura , ADN/ultraestructura , Eritrocitos/ultraestructura , Grabado por Congelación/métodos , Inmunoglobulina M/ultraestructura , Microscopía de Fuerza Atómica/instrumentación , Microscopía de Fuerza Atómica/métodos , Miosinas/ultraestructura , Orthomyxoviridae/ultraestructura , Conejos , Cloruro de Sodio/química

RESUMEN

The collectins are carbohydrate binding proteins which, like C1q, contain collagen-like sequences. The collectins belong to group III of the family of lectins containing C-type carbohydrate recognition domains (CRDs). The structural similarity between the collectins and C1q is clearly demonstrated by electron microscopy in that they all contain multiple polypeptides which are organised into subunits containing triple-helical stalks throughout their collagen-like regions and globular 'heads' in the C-terminal regions. Four, or six, of these structures are associated via distinct, short, N-terminal regions to form the oligomeric molecules seen in the electron microscope. The overall structural similarity between C1q and the collectins, however, does not extend to similarity in amino acid sequences over the C-terminal regions. The C-terminal regions of C1q, unlike those of the collectins, do not contain the conserved residues found in the CRDs present in the C-type lectins. Instead, C1q has a high degree of homology to collagen sequences (Type VIII and X) and this is consistent with the fact that, unlike the collectins, C1q binds to protein motifs in IgG, or IgM, rather than to carbohydrate structures. Also, despite sometimes showing interruptions in their collagen-like regions, the collectins do not always display a 'bend' in their collagen-like 'stalks' similar to that which is seen in C1q. Therefore, C1q may be more closely related to collagens than to the collectins. The collectins can be classed into two distinct group, with MBP and SP-A being hexamers and SP-D, conglutinin and collectin-43 (CL-43) being tetramers, with proteins in the latter group also having significantly larger dimensions with respect to the length of their collagen-like 'stalks'.

Asunto(s)

Proteínas Portadoras/ultraestructura , Complemento C1q/ultraestructura , Lectinas/ultraestructura , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Colectinas , Complemento C1q/química , Glicoproteínas/química , Glicoproteínas/ultraestructura , Humanos , Lectinas/química , Microscopía Electrónica , Modelos Estructurales , Datos de Secuencia Molecular , Proteína D Asociada a Surfactante Pulmonar , Surfactantes Pulmonares/química , Surfactantes Pulmonares/ultraestructura , Seroglobulinas/química , Seroglobulinas/ultraestructura

RESUMEN

Scanning tunnelling microscopy has been used to examine the structure of proteins deposited on a graphite surface. Three molecules have been studied; immunoglobulin G (IgG), Complement component 1q (C1q) and ATP-citrate lyase (ACL). The images show IgG as a tri-lobed molecule, consistent with the known 3D structure as determined by X-ray crystallography. The C1q images differ from the well known "tulip bunch" model derived by electron microscopy, but are consistent with the model if it is assumed that the six globular heads have aggregated. Molecules of ACL are visible as discrete units, with some hints of substructure. These results highlight the potential of STM in studying protein structures, but also illustrate the difficulties of interpreting micrographs of proteins whose structure is currently unknown.

Asunto(s)

Complemento C1q/ultraestructura , Inmunoglobulina G/ultraestructura , Microscopía de Túnel de Rastreo , Modelos Moleculares

RESUMEN

The age of ultrahigh-resolution scanning electron microscopy (SEM) began in 1985, when the UHS-T1, with a resolution of 0.5 nm, was developed. Commercial instruments of the same or similar types followed rapidly. As instrumental resolution progressed, conventional specimen preparation methods became inadequate, and a number of new techniques were devised. In this paper, detailed procedures for these preparation methods such as the CC plate method and heavy metal impregnation are described, together with precautions recommended for achieving ultrahigh-resolution. Some applications of the method to biological specimens are also reported. Morphological identification of immunoglobulins prepared from human blood was attempted, and although the identification was not completely successful this technique may yet come to be of use in the clinical examination of allergic or infectious diseases. SEM images of complement, Clq, proteoglycan and the helical structure of double stranded DNA are shown, as also is the visualization of immunolabelled cell-surface receptors.

Asunto(s)

ADN/ultraestructura , Inmunoglobulinas/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Proteoglicanos/ultraestructura , Carbono , Complemento C1q/ultraestructura , Humanos , Inmunoglobulinas/análisis , Microscopía Electrónica de Rastreo/instrumentación , Receptores de Superficie Celular/ultraestructura

RESUMEN

Previous studies have suggested that C1q reacts with DNA via both the globular region of C1q (GR) and the collagen-like region of C1q (CLR). In this study, the binding of dsDNA and ssDNA to GR and CLR was quantitated by a solid-phase assay. Both dsDNA and ssDNA bound to the GR and CLR of C1q in an ionic strength-dependent manner. Under physiologic salt concentrations, however, dsDNA and ssDNA bound preferentially to CLR and not to GR. The binding of dsDNA to C1q was not affected by heat inactivation of C1q or its exposure to pH 4.45, which abolished the binding of heat-aggregated human IgG (AHG) with C1q. The preincubation of the solid-phase C1q with AHG did not decrease the binding of dsDNA or ssDNA to the solid-phase C1q. These results indicate that the major sites for binding DNA to C1q are located in the CLR of C1q and are not overlapping with those for AHG or immune complexes.

Asunto(s)

Complemento C1q/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Sitios de Unión , Colágeno , Complemento C1q/ultraestructura , Proteínas de Unión al ADN/ultraestructura , Calor , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulinas/metabolismo , Técnicas In Vitro , Fragmentos de Péptidos/metabolismo