Vasileva M.,University of Heidelberg |
Horstmann H.,University of Heidelberg |
Geumann C.,Synaptic Systems |
Gitler D.,Ben - Gurion University of the Negev |
Kuner T.,University of Heidelberg
European Journal of Neuroscience
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 300Hz was accelerated only at frequencies above 100Hz 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. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd. Source
Geumann C.,Max Planck Institute for Biophysical Chemistry |
Gronborg M.,Max Planck Institute for Biophysical Chemistry |
Hellwig M.,Max Planck Institute for Biophysical Chemistry |
Martens H.,Synaptic Systems |
Jahn R.,Max Planck Institute for Biophysical Chemistry
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 78. ng/ml to 77. pg/ml (or 74-0.1. fmol). 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. © 2010 Elsevier Inc. Source
Dahm L.,Max Planck Institute for Experimental Medicine |
Ott C.,Max Planck Institute for Experimental Medicine |
Steiner J.,Otto Von Guericke University of Magdeburg |
Steiner J.,Center for Behavioral Brain science |
And 16 more authors.
Annals of Neurology
Objective We previously reported an unexpectedly high seroprevalence (10%) of N-methyl-D-aspartate-receptor subunit-NR1 (NMDAR1) autoantibodies (AB) in healthy and neuropsychiatrically ill subjects (N = 2,817). This finding challenges an unambiguous causal relationship of serum AB with brain disease. To test whether similar results would be obtained for other brain antigen-directed AB previously connected with pathological conditions, we systematically screened serum samples of 4,236 individuals. Methods Serum samples of healthy (n = 1,703) versus neuropsychiatrically ill subjects (schizophrenia, affective disorders, stroke, Parkinson disease, amyotrophic lateral sclerosis, personality disorder; total n = 2,533) were tested. For analysis based on indirect immunofluorescence, we used biochip mosaics of frozen brain sections (rat, monkey) and transfected HEK293 cells expressing respective recombinant target antigens. Results Seroprevalence of all screened AB was comparable in healthy and ill individuals. None of them, however, reached the abundance of NMDAR1 AB (again 10%; immunoglobulin [Ig] G 1%). Appreciable frequency was noted for AB against amphiphysin (2.0%), ARHGAP26 (1.3%), CASPR2 (0.9%), MOG (0.8%), GAD65 (0.5%), Ma2 (0.5%), Yo (0.4%), and Ma1 (0.4%), with titers and Ig class distribution similar among groups. All other AB were found in ≤0.1% of individuals (anti-AMPAR-1/2, AQP4, CV2, Tr/DNER, DPPX-IF1, GABAR-B1/B2, GAD67, GLRA1b, GRM1, GRM5, Hu, LGl1, recoverin, Ri, ZIC4). The predominant Ig class depended on antigen location, with intracellular epitopes predisposing to IgG (chi-square = 218.91, p = 2.8 × 10-48). Interpretation To conclude, the brain antigen-directed AB tested here are comparably detectable in healthy subjects and the disease groups studied here, thus questioning an upfront pathological role of these serum AB. Ann Neurol 2014;76:82-94 © 2014 American Neurological Association. Source
Agency: Cordis | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2013-ITN | Award Amount: 3.42M | Year: 2013
Even in the simplest cells, the integration of proteins into a biological membrane is a complex process that is frequently coupled to ribosomal protein synthesis, and requires the coordinated actions of several additional cellular machines. The de novo recapitulation of such a complex process is well beyond the scope of our current technical abilities. Indeed, the techniques that are used to create novel proteoliposomes, for drug delivery, and artificial membranes, for synthetic biology, are extremely crude. A common approach is to mix detergent solubilised proteins with lipids, and then remove the detergent to form proteoliposomes, a process that is inefficient and difficult to control. Another major limitation of this approach is our inability to alter the protein complement of the resulting phospholipid-bilayers once they are formed. It is precisely this issue that our consortium will address, by creating a flexible and ubiquitous platform that is ideally suited to incorporating proteins into preformed liposomes. To achieve this novel and innovative breakthrough in liposome technology, we will harness the unusual ability of tail-anchored proteins to be inserted into pre-existing membranes. This technique will enable the production of customised liposomes that can be tailored to optimise drug delivery, and allow the creation of multifunctional artificial membranes for the newly emerging field of synthetic biology. The overriding ethos of our network is to develop a robust platform for the application and exploitation of tail-anchored membrane proteins based on a framework that develops and enhances fundamental insight and training in this new field of research.
Agency: Cordis | Branch: FP7 | Program: CP-IP | Phase: HEALTH-2009-2.2.1-1 | Award Amount: 15.81M | Year: 2010
Signalling at nerve cell synapses - a key determinant of all aspects of brain function - depends on the function of hundreds of synaptic proteins and their interactions. Numerous recent studies showed that a wide range of neurological and psychiatric diseases are synaptopathies whose onset and progression are due to mutations of synaptic proteins and subsequent synaptic dysfunctions. EUROSPIN will pursue a multilevel systems biology approach to determine mechanistic relationships between mutations of synaptic proteins and neurological and psychiatric diseases, and to develop new diagnostic tools and therapies. Our concept is based on the current knowledge of disease genes, which we will continuously extend with new human genetic data and complement with large-scale screens of mutant mice in order to identify and characterize disease-relevant mutations in synaptic proteins and corresponding mouse models. Proteomic tools will be used to analyse the protein components of synapses, and protein interaction networks of synaptic disease gene products will be mapped systematically. In parallel, smart libraries will be employed to develop small molecules for perturbing the functions and interactions of disease gene products. Functional models of disease-relevant protein networks will be generated and used to formulate hypotheses as to how specific mutations might affect synaptic physiology and network function, and thus cause disease. These hypotheses will initially be tested in reduced systems by novel physiological and imaging methods. Well-validated disease gene products, the consequences of their dysfunction in disease, and therapeutic modifications of their dysfunction will then be studied in mouse models in vivo, applying novel electrophysiological, imaging, and behavioural techniques. The combined information obtained in the EUROSPIN program will be used for the development of new diagnostic tools and therapeutic interventions that can be tested in patients.