National project
Biophysical neuroprofiling on experimental models of CNS damage and recovery
Project ID : 143054B
Period : 2006-2010
Principal investigator : prof. Pavle R. Andjus, PhD
Description : Experimental models of CNS damage will be studied through the employment and development of modern biophysical techniques - laser scanning confocal microscopy, electrophysiology ("patch-clamp") and in vivo NMR . These techniques in synergy will be used to reach the basic conditions for the formation of neurophysiological and biophysical profiles - «neuroprofiles» of cells and tissues in the state of unbalanced homeostasis. The studied models will include:
- amyotrophic lateral sclerosis (ALS) - primary cell cultures of neurones and/or glia treated with purified fractions of patient CSF and sera, as well as rats with mutant CuZn superoxide dismutase;
- experimental allergic encephalomyelitis a model of multiple sclerosis - will also serve for a comparative study (vs. ALS) of underlying autoimmune mechanisms;
- transgenic mice deficient in the proteoglycan tenascin - for the study of molecular mechanisms of synaptic restoration.
The expected results will put light on the role of postsynaptic receptors and glutamate transporters, on the significance of oxidative stress and calcium homeostasis, on the intercellular connections of neurons and glia with emphasis to the role of glia, in the processes of injury, healing, and recovery. Neuroprofiling will be used to define inflammatory lesions. Neuroprofiles should facilitate a closer definition of mechanisms of disease and repair, more precise and specific diagnostics, and a more reliable introduction of therapeutic agents.
International projects
"NEUROIMAGE" EU - FP6-INCO WBC SSA3
Project ID : 026400
Period : 2006-2009
Principal investigator : prof. Pavle R. Andjus, PhD
Research objectives addressed :
- to improve the research capacities of West Balkan centres, in the priorities of FP6
- to stabilise and to reinforce the Western Balkan Countries research potential
- to contribute to sustainable research development
The main objective of the project is to improve research capacities of a Western Balkan (WB) research centre and of WBs in general, for the groundbreaking research field of cell and tissue imaging in life sciences for health, particularly neurosciences and neurology. The WB centre, which is the single proposer of this action, will be the Institute for Physiology and Biochemistry, School of Biology, University of Belgrade with its Centre for laser microscopy. In addition to diverse resources for neuroscience and physiology, the Institute is equipped with a laser scanning confocal microscope that will present the core facility for the project research management. The principal objective will be met through a work plan designed to:
- develop the confocal imaging core platform taking into account specific scientific directions, i.e. physiology time-series and plasticity. development and molecular genetics,
- increase the capacity to participate in the research programmes of the EU and to improve already existing links with well-established European research centres via intense networking,
- strengthen the technical capacity by upgrading and maintenance of equipment and by provision of key skills, employment of young researchers and exchange visits of scientists,
- bridge the gap between theoretical and applied science and serve the socio-economic needs of the country by supporting medical and pharmaceutical research through interaction with medical institutions,
- disseminate knowledge of cell imaging beyond the field of neuroscience and forming a wide regional database of potential users and collaborators.
Participants :
- Laboratory of Neuroendocrinology - Molecular Cell Physiology (LNMCP), Institute of Pathophysiology, Medical Faculty, University of Ljubljana, Slovenia
- Institute of Neuroscience CNR (INCNR), Pisa, Italy
- Laboratory of Molecular Neuroscience (LMN), Centre for Molecular Biology and Neuroscience, University of Oslo, Norway
- Centre for Brain Repair “Rita Levi Montalcini” (CRLM) Department of Neuroscience, University of Turin, Italy
- The Croatian Institute for Brain Research (CIBR), School of Medicine, University of Zagreb, Croatia
- The Centre for Laser Microscopy, Institute for Physiology and Biochemistry, University of Belgrade, Serbia
Bilateral projects
Pathogenesis mechanism of C9ORF72 repeat expansion in amyotrophic lateral sclerosis and frontotemporal dementia
Period : 2014-2015
Description : Recently, an ALS and FTLD-associated expansion of the intronic hexanucleotide repeat GGGGCC (G4C2) in the C9orf72 gene was identified, adding these diseases to a growing list of disorders of unstable repeat expansion and further supporting the concept of ALS-FTLD continuum. C9orf72 expansion has been associated with approximately 40% of familial ALS, 25% of familial FTLD and 6-7% of sporadic ALS and FTLD cases, representing the biggest single genetic cause within the TDP-43 proteinopathies. It is currently unknown how C9orf72 repeat expansions causes ALS and FTLD.
With this joint project we aim to elucidate molecular mechanism underlying C9orf72-linked ALS and FTLD, which is one of the most up-to-date topic in ALS field. Advances in our understanding of the molecular pathology in these diseases present an exciting opportunity to identify novel molecular ways of early disease recognition and potentially develop more effective disease interventions.
Participants : The Jožef Stefan Institute, Ljubljna, Slovenia || CLM, Faculty of Biology, University of Belgrade, Serbia
Molecular imaging on cells from animal model of amyotrofic lateral sclerosis - ALSIMAGE
Period : 2010-2011
Description : Method development for fluorescent probes interaction (FRET) in confocal microscopy
Participants : University of Debrecen, Hungary || CLM, Faculty of Biology, University of Belgrade, Serbia
The state of mitochondria in the pyramidal cells of hippocampus after an ischemic episode
Period : 2008-2012
Description : To determine the state of mitochondria in hippocampus following transient global ischemia, the temporal profiles of morphological changes will be studied in the CA1 and CA3 region with light and electron microscopy (EM) up to 7 days following 10 min ischemia in gerbils. Cellular physiology especially related to ion and ROS imaging will also be followed on dissociated cells and tissue systems – mixed neuronal cultures and tissue slices from the rat hippocampus under conditions of ischemia in vitro.
Participants : Academy of Sciences Ukraine || The Serbian Academy of Sciences and Arts
Key personnel :
- Academician Dragoslav Marinković (coordinator) – Serbian Academy of Sciences and Arts
- Prof. Pavle Andjus, Head of Center for Laser Microscopy, Department of Physiology and Biochemistry, Faculty of Biology, University of Belgrade
- Academician Platon G. Kostyuk (coordinator) Ukranian Academy of Scences
- Academician Oleg Krishtal - Ukranian Academy of Scences
- Prof. Galina Skibo, Head of Department of Cytology, Bogomoletz Institute of Physiology of the Academy of Sciences of Ukraine
The role of endosomes in excitotoxic neural damage - the implications in pathogenesis of amiotrophic lateral sclerosis
Period : 2008-2010
Description : Improvement of existing joint research activities, with particular focus on the relationship between endosomes, membrane receptors and ALS will open new possibilities for long-term cooperations and further topics association between national and international projects of both parties, in order to study basic biological mechanisms in the pathogenesis of ALS.
Participants : Croatian Institute for Brain Research School of Medicine University of Zagreb, Croatia || CLM, Faculty of Biology, University of Belgrade, Serbia
Calcium stores in ALS neuroimmunology (IMUNOCALS)
Period : 2009-2010
Description : The main objectives of this collaboration will be:
- To obtain IgGs and their sub-fractions from patients suffering from (a) familial and (b) sporadic ALS, at different controlled disease stages, (c) from healthy donors and other neuropathologies without immune compromise (e.g., Parkinson’s or Alzheimer’s diseases), and (d) compare these fractionated samples with those obtained from the rat ALS G93A model.
- To systematically evaluate the effect of ALS IgGs on intracellular Ca2+ and to connect the results to the function, and release of lysosomes as the recently suggested main release pool in astrocytes (Li et al J Neurosci. 28:7648-58, 2008).
- To study the effect of ALS IgGs on the balance among different acidic Ca2+ stores, endocytosis, and the ER known to operate with multiple Ca2+-releasing messengers. This will be studied on different well known cell models currently used in the CNRS-lab, such as pancreatic acinar cell and other relevant cell lines in culture (Menteyne et al. Current Biology 16:1931–1937, 2006)
- A major aim is to train Belgrade personnel on the advanced electrophysiology and Ca2+-imaging tools developed in the CNRS lab so as to allow the technology transfer. For the CNRS laboratory, the chief interest is to evaluate a new paradigm of ALS that could provide a functional link between lysosomal Ca2+ pool, exocytosis and neurodegenerative pathology.
Participants : CNRS-MSEP, France || CLM, Faculty of Biology, University of Belgrade, Serbia
Neuroimmunology of ALS - a biophysical approach
Period : 2007-2009
Description : Sporadic and familial ALS (amyotrophic lateral sclerosis) differ in the aetiopathogenesis although they have similar clinical symptoms and neuropathology. Moreover, there is substantial evidence that both types of ALS develop an oxidative stress phase (by extensive glutamate stimulated Ca2+ inflow or by impaired SOD activity, or by both being interconnected) that eventually leads to excitotoxicity. We will test if this putative mutual path towards excitotoxicity also comprises autoimmune mechanisms, i.e. if Igs from familial ALS patients resemble Igs from sporadic ALS cases in their proven ability to cause facilitation of excitatory postsynaptic activity (observed in whole cell patch- clamp). To this effect primary cell cultures from wild type and an transgenic SOD1 G93A mutant rat model of familial ALS (already maintained in Belgrade) will be employed. Thus, the effect of ALS Igs will be compared to the specific behaviour in SOD1 G93A mutant cells.
There is substantial evidence that glutamate transport could be affected in sporadic as well as in familial ALS. Glutamate transporters may be another possible target of ALS Igs or of oxidative stress in SOD1 G93 mutants. To test this hypothesis we will study the effect of KCl stimulation prior to or after ALS Igs application as well as in conditions of experimentally impaired neuronal or glial glutamate transport in wild type and transgenic animals.
The obtained results may offer basic protocols for early neuroimmunological in vitro diagnostics of ALS. In addition, by means of short-term visits and workshops the grant will offer training of young researchers in confocal microscopy and calcium imaging.
Participants : CNRS-MSEP, France || CLM, Faculty of Biology, University of Belgrade, Serbia
Multilateral projects (actions)
Academic synapsing in the Balkans
Period : 2012-2014
Type : Multilateral project with Germany (DAAD project "Akademischer Neuaufbau Südosteuropa")
Description : Education of MSc and PhD students - experimental methods in neurosciences
Participants : University of Podgorica, Montenegro || Saints Cyril and Methodius, University of Skopje, Republic of Macedonia || University of Leipzig, Germany || CLM, Faculty of Biology, University of Belgrade, Serbia
Brain Extracellular Matrix in Health and Disease (ECMNet)
Period : 2010-2014
Type : COST Action BM1001
Description : The objective of this Action is to understand the rapidly emerging role of the neural extracellular matrix (ECM) in brain function and dysfunction, by synergising European research in this multidisciplinary domain. The synergy is essential to study complex interaction between ECM, neurons and glial cells, which have historically represented separate fields of science. This Action will be instrumental to address the following scientific questions: what are the functional properties of different ECM components in the brain, which new research tools are in need to study neural ECM, which ECM molecules are linked to brain disorders, which drugs can target them, and how to synthesize three dimensional ECM scaffolds that would mimic specific forms of neural ECM and guide reconstitution of neural circuits.The future economic/societal benefits from this Action will be development of novel ECM-based therapeutic strategies for major neurological, neurodegenerative and neuropsychiatric disorders, such as epilepsy, traumatic brain injury, dementia, schizophrenia, and mental retardation.
Link : COST Action BM1001
“Akademischer Neuaufbau Südosteuropa (Stabilitätspakt SOE)
Period : 2010-2011
Type : Multilateral project with Germany, Deutsche Akademische Austausch Dienst (DAAD)
Description : The goal of this project is to establish a cross-regional cooperation between the Academic Institutions in Belgrade-Serbia, Kotor-Montenegro and Leipzig-Germany. This initial endeavor should provide a basis for future scientific and academic projects aiming at better qualification of undergraduate and graduate students. Moreover, the mutual research interests in the area of neurobiology (elucidated in the proposed research project) are expected to result in joint international high-impact research projects in the future.
In the scope of the recent implementation of the Bologna Process, this project enables excessive academic exchange between the professors and students from Belgrade, Kotor and Leipzig and thereby offers good insights into European academic education standards with different backgrounds. By visiting laboratories in partner institutions, master and graduate students shall obtain broad spectrum knowledge about the experimental approaches in neuroscience. This will be of great importance for their professional development and future perspectives.
Neural Regeneration and Plasticity (NEREPLAS)
Period : 2005-2010
Type : COST Action B30
Description : The main objective of this Action is to increase the knowledge on the neuronal processes underlying functional recovery following traumatic, ischemic or degenerative damage of the nervous system, and their relationship with those neural processes involved in motor and cognitive learning.
Link : COST Action B30
Grants
Verification of trace elements on the subcellular level in astrocytes isolated from a rat model of Amyotrophic Lateral Sclerosis
Year : 2015
Type : Swiss Lightsource (SLS), Paul Scherrer Institut (PSI), Villigen, Switzerland
Description : We aim to use synchrotron x-ray fluorescence (XRF) imaging and nano-spectroscopy to assess the trace element profiles, especially Cu and Zn, as well as complete metal distribution within the neural cells isolated from a rat model of Amyotrophic Lateral Sclerosis (ALS) containing the mutated Cu/Zn superoxide dismutase protein (SOD1 G93A). Here we propose to use novel nano-setup for the XRF in order to compare trace elements on the subcellular level in the astrocytes, neural cells isolated from the SOD1 G93A rat model of ALS and control animals. Detailed examination of metal ion homeostasis and its role in SOD1 aggregation, which is the pathological hallmark of the disease, within astrocytes in ALS has not been performed before and therefore little is known about the changes in astrocyte intracellular ion composition occurring in this ALS model.
SOD1 protein aggregates in intact astrocytes – a cellular model of Amyotrophic lateral sclerosis
Year : 2015
Type : recording at CELLS - ALBA Synchrotron, beam line BL09 - MISTRAL, Barcelona, Spain
Description : Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known, however the interplay between motor neurons and astrocytes is crucial in the outcome of the disease. In this proposal we intend to use soft x-ray tomography in the so-called “water window” energy range at the beamline Mistral to investigate the SOD1 protein aggregation. The goal is to make the first set of experiments on an in vivo system, in order to investigate intracellular protein aggregates that play a key role in ALS as well as in other neurodegenerative proteinopathies. So far the nucleation and structuring of aggregates was not studied in detail.
To this aim we will study cultured astrocytes from the SOD1 G93A ALS rat model with the overexpressed mutant copper-zinc superoxide dismutase (SOD1). We have initially demonstrated that astrocytes that generally have many important functions in maintaining and nourishing the central nervous system neurons, in ALS express increased levels of protein aggregates. The expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread. The first experimental step would be to test the feasibility at the soft x-ray microscope at the beamline Mistral, ALBA, to investigate in situ protein aggregates targeted with the immuno-gold in the intact cryopreserved cells. We intend to precisely localize and structurally analyze the nuclei of intracellular protein aggregation in intact astrocytes aiming to understand the beginning of the formation of these biomolecular disease markers and the development of their structure-function relationship. This may finally offer therapeutic solutions and time windows to overcome mutant SOD1 cytotoxicity.
ERAWEB - Erasmus Mundus-Western Balkans
Year : 2013
Objectives : Reinforcing academic and research collaboration for the benefit of teaching and research as well opening of a new channel for exchange of students and for mutual MSc and PhD theses.
Host : Università Cattolica del Sacro Cuore, School of Medicine "A. Gemelli"
"Centre of Excellence" grant
Period : 2006-2008
Awarded by : Psysiological Society (UK)