Aim: To understand structure - function relationships of muscle and neuronal AChRs with ultimate aim the elucidation of their pathogenic mechanisms and the development of novel therapies.

We are expressing extracellular domains of human muscle and neuronal AChRs, as well as intact AChRs, and we are studying their structure and their interaction with specific ligands and antibodies. Specifically, we perform:

a. Heterologous expression of the extracellular domains of α1, α4, α7, β1, β2, γ, δ,  and ε AChR subunits and coexpression of subunit combinations, in eukaryotic expression systems.
b. Heterologous expression of almost the whole AChRs, by deletion of only their flexible cytoplasmic domains, in order to facilitate their crystallization.

c. Design and construction of mutant forms of these molecules with improved structural and functional properties (in collaboration with Prof. E. Eliopoulos, Agricultural University of Athens).
d. Mass production and purification of the recombinant proteins.

e. Production of monoclonal antibodies against these molecules.

f. Ligand binding studies.

g. Preliminary structural studies (by DLS and CD spectroscopy).

f. Preparation of the above molecules for NMR studies in collaboration with Dr. George Spyroulias, University of Patras.
e. Crystallization of extracellular domains from several AChR subunits and their complexes with ligands and antibodies. This part was initiated in collaboration with the group of the late Nikos Oikonomakos, NHRF, Athens, an eminent crystallographer and excellent personality.
We have determined the conditions for crystallization of two AChR subunits some of which diffract X-rays. This is essential for the determination of their structure; however, additional improvements are required. We have recently acquired a robotic system (Oryx4, Douglas Instruments) for automated crystallization trials, which is expected to greatly increase our rate of progress.

We ultimately aim to map in detail the receptor ligand binding sites (such as for acetylcholine, nicotine, etc) through the analysis of receptor-ligand complexes, which will eventually lead to the rational design of novel pharmaceuticals against neurological disorders.

B. TOWARDS UNDERSTANDING MYASTHENIA GRAVIS (MG)

Aim: Study the mode of action of the pathogenic MG autoantibodies and development of an antigen-specific therapy for MG.
As it is described in the introduction, myasthenia gravis (MG) is caused by autoantibodies against the AChR (usually) or MuSK (occasionally), both located at the post-synaptic membrane of the neuromuscular junction.

1. Antigen-specific therapeutic removal of pathogenic antibodies

We aim at the selective removal of the anti-AChR and anti-MuSK autoantibodies of MG patients’ blood by the use of columns carrying extracellular domains of muscle AChR subunits or MuSK, capable of selectively binding these antibodies.  Prerequisite for this aim is the heterologous expression of large quantities of these protein domains with conformation appropriate for binding the corresponding autoantibodies. Specifically, we perform:

• Heterologous expression of the extracellular domains of all five muscle AChR subunits and of MuSK in eukaryotic expression systems (yeast Pichia pastoris and insect cells)
• Construction of mutant forms of these recombinant proteins with improved antigenic characteristics.
• Mass production and purification of the recombinant proteins. Preparation of immunoaffinity columns on sepharose beads.
• In vitro studies: testing the ability of the immunoaffinity columns to safely and efficiently adsorb the anti-AChR and anti-MuSK antibodies from large volumes of MG plasma or blood.
• Animalstudies: Application of the immunoadsorption in experimental animal models.
• Design of future application of the immunoadsorption in clinical trials.

This effort is already at an advanced preclinical stage, it has lead to several publications and to a patent, and we hope to be able to initiate clinical trials before long.

2. Investigation of the functional mechanisms of autoantibodies in MG and search for responsible genetic factors

Autoantibodies against the AChR or MuSK are isolated from MG patients’ sera, as well as other serum components with potentially pathogenic or protective function, and their role is studied in cell culture and animal models.

Additionally, we are screening patients’ blood for single nucleotide polymorphisms (SNPs) in genes involved in the regulation of the immune response, which may be linked to predisposition of developing MG (in collaboration with Ass. Prof. G. Patrinos, Department of Pharmacy, University of Patras).

C. AQUAPORIN-4 AND NEUROMYELITIS OPTICA (NMO)

Taking advantage of our experience with MG, we have recently initiated the study of another autoimmune disease, neuromyelitis optica (NMO), or Davic’s disease, which is related to multiple sclerosis. NMO is believed to be caused by autoantibodies against the water channel, aquaporin-4, in glia cells of the brain. Our initial aim is to explore the pathogenic role of the autoantibodies against aquaporin-4 in experimental animals. We have expressed aquaporin-4 in eukaryotic expression systems and using affinity chromatography we are isolating the specific autoantibodies from NMO patients’ sera, which will then be used in animal studies (development of experimental animal models).

D. DEVELOPMENT OF ULTRA-SENSITIVE DIAGNOSIS FOR AUTOIMMUNE NEUROLOGICAL DISEASES

In addition to the routine tests, which are already used in our lab to diagnose three neurological diseases, MG, NMO and LEMS (i.e. detections of autoantibodies against AChR, MuSK, titin, aquaporin-4 and calcium channels), we are developing much more sensitive, than the currently available, techniques for the detection of very small quantities of the pathogenic antibodies against these antigens in order to reduce the chances for false negative diagnoses. We are already applying the ultra-sensitive technique for the routine diagnosis of anti-aquaporin-4 antibodies and we are working on the establishment of similar techniques for the anti-AChR and anti-MuSK antibodies.

Collaboration with Greek laboratories/Institutes

In addition to the close collaboration with the Molecular Biology and Immunology laboratory of the Department of Pharmacy, University of Patras, we collaborate with the groups of:

• Prof. E. Eliopoulos, Agricultural University of Athens.
• Ass. Prof. E. Grapsa, Alexandra Hospital, Athens.
• Prof. K. Iatrou, Demokritos Institute, Athens.
• Dr. D. Leonidas, NHRF, Athens.
• Dr. Μ. Roberts, Biomedical Research Foundation, Athens.
• Associate Prof. G. Spiroulias, University of Patras, Department of Pharmacy.

Collaboration with international laboratories/institutes

- Prof. M. De Baets, Faculty of Medicine, Univ. Limburg, Maastricht, The Netherlands.
- Dr. S. Berrih-Aknin, Hopital Marie Lannelongue, Le Plessis Robinson, France.
- Prof. L. Chen, Univ. of Southern California, Los Angeles, USA.
- Prof. P. Christadoss, University of Texas Medical Branch, Galveston, Texas, USA
- Prof. S. Fuchs, Dep. of Immunology, Weizmann Institute of Science, Rehovot, Israel.
- Dr. M. Skok, Bogomoletz Institute of Physiology, Kiev, Ukraine.
- Pro. T. Sixma, Netherlands Cancer Institute, Amsterdam, The Netherlands
- Prof. A. Smit, Vrije University, Amsterdam, The Netherlands
- Prof. V. Tsetlin, Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.
- Dr. N. Unwin, Molecular Biology Laboratory, MRC, Cambridge, UK.
- Prof. A. Vincent, Univ. of Oxford, Inst. Molecular Medicine, Oxford UK.
- Prof. W. Welte, Univ. Konstanz, Konstanz, Germany.