The Gert Lubec Proteomics Laboratory

Lab members

Gert Lubec

• Full professor for Neuroproteomics at the Medical University of Vienna
• Editor-in-chief of Amino Acids, the forum for Amino Acid, peptide and protein research
• Member of the editorial board of Proteomics and in top neuroscience journals
• Job description - Generating project ideas and concepts, supervision of research work and writing the manuscripts.

For Neuroscience Division

• Generation of project main ideas and concepts.
• Setting short term and long term aims.
• Control performance of the process.
• Active listening of the feedback from junior scientists.
• Trouble shooting and supervision.

Deamidation could be a rapid and effective way to inactivate brain receptors and thus represent a new tentative regulation system. Neurobiologically, a link between receptor regulation by deamidation and L&M would be interesting and is studied by N.B. After confirmatory results using a genetically deamidase deficient mouse strain the possible role of deamidation will be studied in different forms of L&M.

Beto is interested in spider webs and spider silk glands and this is a great challenge indeed because the web consists of several proteins that are difficult to analyse. Different parts contain different proteins, glues and of course neither cross-linking nor glue proteins are unambiguously identified nor are the posttranslational modifications. Do spiders show the same posttranslational modifications as in the mammalian system? What is the structure and what are the mechanoelastic properties? Applications of this strongest material may arise in the future both, in industry and medicine.

Soheil is interested in mechanisms of cognitive enhancement in terms of the concerted action of receptors in several areas of the brain.

Looking for a cognitive enhancer that works in landmazes is followed by the concomitant determination of receptor stoichiometry and cross talk, between receptors and between areas. Cognitive enhancement will focus on cholinergic stimulation that in turn would be tested in spatial, olfactory and contextual memory paradigms. What happens to NMDA, AMPA, GABA_A and GABA_B and serotonin receptors when the cholinergic system is activated and leads to cognitive enhancement?

Claudia is interested in the neurobiology, neurochemistry and pharmacology of jealousy in rodents. Jealousy (J) can be seen as a physiological trait to protect a relationship, sexual or non-sexual in nature, including sibling rivalry and friendship. It may be, however, considered a symptom of several psychiatric diseases or even a disease per se, morbid jealousy or delusional jealousy. Jealousy may be a symptom of several psychotic entities including schizophrenia and obsessive-compulsive disorder (DSM-III-R). Moreover, alcoholism is associated with high J incidence. Jealousy may be a serious issue as a complex affect destroying normal social life and indeed, J has been reported to be a major underlying cause of partner violence and homicide (uxoricide). Reports on antipsychotic pharmaceutical treatment of J are anecdotal.

With the exception of the description of the jealousy of a dog in Science 1892 no animal studies were published so far and mainly phenomenological descriptive work is available in humans. No studies on pathophysiology, neuroanatomical or electrophysiological changes or neurochemistry have been carried out in animals and there is no animal model available so far. J is, however, of main interest in forensic medicine, psychology and psychiatry and thus represents a major challenge to approach this problem in basic neurosciences.

Animal models for induction of sexual and non-sexual jealousy will be created forming the basis for all following studies. The area(s) involved in the induction of J will be identified by immunohistochemical studies using c-foc as an indicator of brain area activation. The corresponding areas will be punched out and used for biochemical studies determining receptors and their ligands as well as brain specific protein expression. In parallel, microdialysis studies to examine neurotransmitter systems (aminergic, cholinergic, serotoninergic, GABAergic, glutamatergic and peptidergic) will be carried out in the areas identified as J - related.

Functional studies using animals with specific deficits will serve as controls for the findings above. Biological function of proteins with differential levels or posttranslational modifications in the corresponding areas will be tested in animal models.

Pharmacological treatment based upon the findings from above will be established and evaluated in the proposed animal model.

- hypothesis 1, jealousy can be induced in animals - hypothesis 2, jealousy involves brain regions known to include areas relevant for affects including hypothalamus, the limbic system, the septum, frontal cortex, prefrontal cortex - hypothesis 3, jealousy involves the aminergic system, in particular dopamine and serotonin - hypothesis 4, animals with jealousy will respond to corresponding antipsychotic treatment - hypothesis 5, protein derangements and posttranslational modifications will occur in thecorresponding regions and will provide insight into cascades and pathways involved in jealousy

A series of receptors has been shown to be involved in memory; Maryam is interested in the concerted action of major receptors as cholinergic, serotonine 1A, GABA_A and GABA_B, AMPA and NMDA receptors. Following the probe trial in the multiple T-maze the receptor systems will be extracted from hippocampus and cortical areas in the C57BL/6J mouse and a gel-based system is used to determine receptor proteins and post-translational modifications. The effect of protein level changes and PTM will be tested electrophysiologically. Post-translational modifications of interest will be pharmacologically modified in vivo and in vitro and MTM experiments re-run.

The serotonin receptor 1A is a key element in neural transmission and signaling. This membrane protein plays a central role in L&M as well as mood states. As so far only significant work was carried out by immunochemical techniques, work at the protein rather than the immunochemical level is pivotal. S.H. is about to fully characterise the 5/HT1A/R and its posttranslational modifications from mouse hippocampus. This will be forming the basis for all further serotonin/related work because so far only 5-HT1A-R immunoreactivity can be determined. The aim is therefore to characterise 5-HT1A and other serotonin rceptors, their splice variants and PTMs and to reveal the role of these receptors in L&M in our paradigms in the mouse.

The 5-HT1A-R will be isolated using our native gel systems and conformational studies will be carried out and attempts to assemble the receptor unit to a functional unit will be an ambitious and major promising goal.

Drebrin is an acting bundling protein thus forming dendritic spines. A drebrin A knock-out mouse was generated and is now under neurophenotyping in our laboratory. Drebrin-dependent protein levels and in particular receptor proteins in different areas will be determined and linked to the proposed L&M and other behavioral deficits using several paradigms.

Based upon these results histochemical, electronmicroscopical and immunohistochemical studies will be carried out to show the cell types and subareas and hippocampal slices as well as hippocampal neuronal cultures will be used to provide the electrophysiological correlates. Drebrin A and splice variants will be identified and fully characterised by gel/based mass spectrometry that would include detection of PTMs. Results will indicate whether drebrin E, the embryonic form of the protein or a probable third splice variant, detected by immunoblotting, will be able to compensate for drebrin A deficiency.

As preliminary studies point to anxiety/related behavior, proteins from the corresponding areas will be analysed as abovementioned.

Treatment of the potentially occurring deficits as e.g. Anxiety related behavior will be treated by corresponding medication and mice are re/analyzed by the same methods.

Phosphorylation is a major event in the formation of L&M and a large amount of protein kinases and phosphatases have been described. In the hunt for kinases we identified 35 (out of 500) kinases in hippocampus, that were linked to spatial memory formation in the mouse. These are being verified by immunochemical techniques and confocal microscopy. These kinases are now identified in several areas of the brain linked to memory formation and storage. The cross-talk between these kinases is a major goal in this project. Electrophysiology is being employed to verify the neurobiological relevance of kinase levels-activity. Modification of kinase levels within the different hippocampal subareas and different neurons using specific virus promoter transfection may assist to specify results. Corresponding in vivo studies are carried out in parallel.

Edit is interested in signaling networks in Alzheimer´s disease. Complementing previous work on neuroproteomics of AD brains the focus should be set at brain receptors and kinases. And indeed, no relevant work was carried out so far on this subject and working with human brain samples was almost impossible due to the long post-mortem intervals. This problem was solved by having obtained valuable samples from a brain bank with low PM intervals. In addition, own analytical work enables determination of all known brain receptors including those indispensable for memory formation and this also holds for kinases. Fishing kinases deranged in AD will be used for investigating into the function of these kinases and will be pharmacologically and genetically manipulated and the in vitro and in vivo relevance of blockades will beperformed at the morphological, electrophysiological and proteomic level.

There is still no convincing treatment for AD and our approaches may be contribute to solve this problem affecting millions of people worldwide.

Sonia is interested in the evolutionary question why a butterfly and a sea snail are sharing a proteodoxin!!! So far there is no explanation for this phenomenon and the approach of our group is now to chek this toxin in a large series of different ordines and families. Gel-based proteomics studies as well as immunocytochemical analyses have to be carried out on large scale. At the same time the biogeographical distribution of this toxin is of eminent interest and basic principles in developmental and evolution biology may be answered.

Cognitive enhancers (CE) are still holding centre stage in academia and industry.

A series of mechanisms have been proposed and cognitive enhancers are from several chemical compound classes. Unassigned is focussing on finding new CEs as well as studying mechanisms of action in terms of protein pathways and cascades for known ones. In a systematic approach new compounds obtained from cooperations are tested in spatial memory paradigms and hydrophilic and hydrophobic brain proteins are being linked to improvement of spatial memory.

When successful in spatial memory these compounds are also tested in fear conditioning and olfactory memory. It is expected that protein pathways utilized for CE are comparable between different memories. CE is studied at the protein level and in terms of PTM but are verified by electrophysiological and immunohistochemical methods and genetically manipulated animal systems and siRNA inhibition.

Effective CEs finally are tested in animals with poor memory and in animal models of cognitive disorders.

Generation of protein networks in memory formation, storage and thinking in the rodent

Sunetra is interested in the generation of novel protein networks that have not been described so far in memory pathways. Based upon own shotgun proteomics work enriching and quantifying phosphopeptides in hippocampi of mice, trained and untrained, in a landmaze, a series of proteins were detected that would be of pivotal interest and have not been linked so far to memory. Moreover, a paradigm testing thinking-like behavior in rodents will be used to determine protein networks in this cognitive system.

Abovementioned own results showed a series of phosphopeptides of proteins with unknown function, hypothetical proteins and novel proteins in addition to well-described proteins. Involvement in cognition would assign a function to these proteins.

Pathway analyses will reveal interactomes and the networks in different forms of memory (spatial, contextual and olfactory) and it will be shown if different forms of memory are employing the same pathways and cascades.

Silencing of the differentially expressed proteins will confirm protein functions in memory and thinking-like behavior as evaluated by in vivo experiments and electrophysiology.

Although aging and memory has been studied from several aspects, the proteomic approach to differentiate hippocampal protein levels between developmental stages has not been systematically studied so far. Several mouse strains are used at different ages and these are compared to each other. Linkage between hippocampal protein levels, splice variants, ptms and parameters from different memory paradigms is being assessed. Animals with good and bad memory performance will be identified and differences between protein levels in several memory-related areas will be examined.

Last not least, Siva will treat animals of different ages with CE s that were effective in own previous studies. We expect that when treated animals with CEs reach memory levels of the animals with good performance of L&M, their protein levels in hipocampus and other areas will be comparable.

Immunchemical, histochemical and electrophysiological tests will complete the findings.

Fernando is interested in the analysis of post-translational modifications (PTM) of proteins. So far analysis of PTMs are relying on mass spectrometry data and this is justified because of the determination of the position of a given PTM. But still, there is only a mass shift provided and verification by another analytical principle is mandatory. But also sample preparation for the detection of PTMs has to be adapted to the nature of the PTM: many PTMs cannot be detected due to cleavage by analytical steps as e.g. on 2DE. HPLC, immunochemical methods are already in use and it is the major goal to develop pure methods from organic chemistry to verify the individual PTMs. Moreover, specific reactions of organic chemicals may allow quantification of the PTM.

Solving this basic problem would further proteomic studies in biology and disease.

Long-term potentiation is a cellular model of learning and memory and is divided into two basic phases, a protein synthesis-independent early phase and a protein synthesis-dependent, late phase.

Induction of hippocampal LTP mainly depends on activation of kinases, phosphatases and proteases but involvement of protein modifications per se have not been systematically addressed so far.

Wanjia is interested in the model of tractus perforans-area dentata-synapse of the rat answering these questions. LTP will be induced in vivo stimulating the right medial tractus perforans with recordings in dorsal hippocampus. Following establishment of LTP in the freely moving rats, dorsal hippocampus will be used for gel-based proteomics studies testing receptors, kinases, phosphatases and their post-translational modifications. This may result into identification of mechanisms for memory formation at the electrophysiological level and pharmacological targets.

Tissue samples are divided into aliquots because receptor proteins will be handled in the native state. Membrane proteins and cytosolic proteins will be extracted by previously published methods (Nature Protocols). In particular receptor proteins will be extracted in the native state and run in the native state using blue native gel electrophoresis. Western blotting using receptor protein antibodies against receptors known to be involved in memory formation will be carried out. Membrane proteins along with the receptor proteins will be run on three dimensional gel electrophoresis and will be unambiguously identified. Aliquots will be also kept to verify mass spectrometry results by immunoblotting. In addition to receptor proteins and other membrane components kinases will be assayed first by kinexus protein arrays for kinases and then individual kinases will be verified and further tested in electrophysiological settings. Potentially new cascades will be followed by more in-depth methodology. Receptor binding to ligands will be tested by a gel-based radioligand assay on native gels and denaturing gels containing the receptor protein. Posttranslational modifications are key elements in signaling and proteins of interest will be tested for PTMs using most updated software and appropriate technology of LC-ESI-MS/MS using a high performance trap with two dfifferent fragmentation methods, CID and ETD.

Sara is interested in major brain receptor complexes and indeed, it is the receptor complexes that would do the work in the brain. A series of brain receptors will be studied in health and disease but we are about to identify the concerted action among individual receptor complexes from both, pre-and postsynaptic sites. The serotonin receptors will be a focus within this investigation and these receptor complexes will be analysed for the composition because of course protein kinases, phosphatases will participate in the assembly of receptor complexes making up the high diversity of these protein complexes.

Georg is interested in thinking-related proteins. The goal is to find and invent a design that allows evaluation of thinking-like behavior in rodents. First approaches include formation of strategies and decision making but higher cognitive tests are still to be designed. As soon as these are created, c-fos and in vivo morphological studies will identify the areas required for thinking-related behavior. Following identification of areas involved, receptors will be characterised mediating this higher cognitive function as well as corresponding signaling cascades