Wirths O.,University Medicine Goettingen |
Erck C.,Synaptic Systems |
Martens H.,Synaptic Systems |
Harmeier A.,Free University of Berlin |
And 11 more authors.
Journal of Biological Chemistry | Year: 2010
N-terminally truncated Aβ peptides starting with pyroglutamate (AβpE3) represent a major fraction of all Aβ peptides in the brain of Alzheimer disease (AD) patients. AβpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aβ. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AβpE3 and studied the potential involvement of oligomeric AβpE3 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 Aβ plaque load and AβpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AβpE3 oligomers. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Agency: European Commission | 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.
Agency: European Commission | 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.
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 | Year: 2014
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.
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 | Year: 2012
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.
PubMed | Uppsala University, Medical University of Vienna, University of Helsinki, Synaptic Systems and University of Gottingen
Type: | Journal: Acta neuropathologica communications | Year: 2016
In Alzheimers disease (AD) a variety of amyloid -peptides (A) are deposited in the form of extracellular diffuse and neuritic plaques (NP), as well as within the vasculature. The generation of A from its precursor, the amyloid precursor protein (APP), is a highly complex procedure that involves subsequent proteolysis of APP by - and -secretases. Brain accumulation of A due to impaired A degradation and/or altered ratios between the different A species produced is believed to play a pivotal role in AD pathogenesis. While the presence of A40 and A42 in vascular and parenchymal amyloid have been subject of extensive studies, the deposition of carboxyterminal truncated A peptides in AD has not received comparable attention. In the current study, we for the first time demonstrate the immunohistochemical localization of A37 and A39 in human sporadic AD (SAD). Our study further included the analysis of familial AD (FAD) cases carrying the APP mutations KM670/671NL, E693G and I716F, as well as a case of the PSEN1 Exon9 mutation. A37 and A39 were found to be widely distributed within the vasculature in the brains of the majority of studied SAD and FAD cases, the latter also presenting considerable amounts of A37 containing NPs. In addition, both peptides were found to be present in extracellular plaques but only scarce within the vasculature in brains of a variety of transgenic AD mouse models. Taken together, our study indicates the importance of C-terminally truncated A in sporadic and familial AD and raises questions about how these species are generated and regulated.
PubMed | Max Planck Institute for Biophysical Chemistry, University of Leipzig, Effigos AG, Synaptic Systems and University of Gottingen
Type: | Journal: Scientific reports | Year: 2016
Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimers disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration.
Tegge W.,Helmholtz Center for Infection Research |
Bonafe C.F.S.,University of Campinas |
Teichmann A.,Helmholtz Center for Infection Research |
Erck C.,Synaptic Systems
International Journal of Peptides | Year: 2010
Side-chain oligo- and polyglutamylation represents an important posttranslational modification in tubulin physiology. The particular number of glutamate units is related to specific regulatory functions. In this work, we present a method for the synthesis of building blocks for the Fmoc synthesis of peptides containing main chain glutamic acid residues that carry side-chain branching with oligo-glutamic acid. The two model peptide sequences CYEEVGVDSVEGEG-E(E x)-EEGEEY and CQDATADEQG-E(E x)-FEEEEGEDEA from the C-termini of mammalian α1- and β1-tubulin, respectively, containing oligo-glutamic acid side-chain branching with lengths of 1 to 5 amino acids were assembled in good yield and purity. The products may lead to the generation of specific antibodies which should be important tools for a more detailed investigation of polyglutamylation processes. © 2010 Werner Tegge et al.
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
Analytical Biochemistry | Year: 2010
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.