Professor Sara Fuchs
Immunotherapy of Myasthenia Gravis
Myasthenia Gravis (MG) and its animal model disease, experimental autoimmune myasthenia gravis (EAMG), are autoimmune diseases in which the nicotinic acetylcholine receptor (AChR) is the target autoantigen. We are interested in the studying the immunological mechanisms and specific regulation of MG. In particular, we are attempting to develop specific immunotherapy for myasthenia.
We have demonstrated that mucosal administration of recombinant fragments corresponding to the extracellular domain of the human AChR -subunit have an immunosuppressive effect on EAMG in rats. Protection against subsequent induction of EAMG and immunosuppression of ongoing disease were accompanied by a reduction in AChR-specific T cell proliferative responses, IL-2 production and anti-self AChR antibodies, and a switch in IgG isotypes, from IgG2 to IgG1. Treated animals exhibit a Th1 to Th2/Th3 shift in their cytokine profile and down-regulation of costimulatory factors. In severely affected rats, this antigen-specific approach may need to be supported by direct modulation of key cytokines and costimulatory factors known to be involved in the pathogenesis of EAMG.
To address this question, we have been employing antibodies either to the proinflammatory cytokine IL-18 or to the costimulatory factor CD40L, to immunomodulate EAMG. These treatments act via different mechanisms but both lead to alleviation of clinical symptoms even when given at the chronic phase of EAMG. Both impaired AChR-specific Th1 cell differentiation with no effect on Th2-type responses. Anti-CD40L administration led to a significant decrease in humoral responses, whereas anti-IL-18 treatment affected mainly cellular responses to AChR. The most significant suppressive effect of both treatments was observed 2-3 weeks after initiation of treatment and was later diminished, implying that blockade of either IL-18 or CD40L alone, may not be sufficient to suppress chronic myasthenia. We suggest that antagonists to key cytokines and/or costimulatory factors be used in conjunction with the antigen-specific treatment, such as induction of mucosal tolerance with AChR recombinant fragments.
The Binding Site of Acetylcholine Receptor (in collaboration with E. Katchalski-Katzir)
Nicotinic acetylcholine receptors (AChR) are pentameric ligand-gated ion channels located in the nervous system and at the neuromuscular junction. Abnormal function of these receptors appears to be involved in several diseases such as Alzheimer's disease, Parkinson's disease, schizophrenia, myasthenia gravis, alcoholism, etc. Molecules that stimulate or block AChRs may be useful in treating such diseases. The ligand-binding domain of these receptors is the ultimate target for such molecules. Thus, the detailed structure of the AChR's binding domain is of utmost importance for drug design.
-Neurotoxins, such as -bungarotoxin (-BTX), bind specifically and with high affinity to AChR and have been most instrumental in the analysis of the ligand binding site of AChR. By using a combinatorial phage display-epitope library we have previously identified a 13-mer peptide mimotope that interacts with -BTX and has similar structural motifs to the the binding region of the AChR -subunit. The three-dimensional solution structure of the complex of -BTX with this library-lead peptide was solved by two-dimensional NMR spectroscopy (in collaboration with Y. Anglister and T. Scherf). The bound peptide was found to adopt a globular conformation, whereas the free peptide in solution was characterized by a rather extended conformation.
Based on this NMR study and on our previous structure-function analysis of the AChR binding site, additional series of 56 peptides, resulting from systematic residue replacement in the lead peptide, one or more replacements at a time, were designed and characterized. Of these, four peptides, designated high affinity peptides (HAPs), homologous to the binding region of AChR, inhibited the binding of -BTX to AChR with IC50 of 2 nM. The solution and crystal structures of complexes of -BTX with HAP, were solved (in collaboration with T. Scerf and J. Sussman, respectively), demonstrating that the HAP fits snugly to -BTX and adopts a -hairpin conformation. Superposition of the X-ray structures of the bound HAP and the homologous loop of the acetylcholine binding protein (AChBP), results in a model indicating that -BTX wraps around the receptor binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits, where it inserts a finger into the ligand-binding site. Our proposed model explains the strong antagonistic activity of -BTX, and accommodates many of the biochemical data on the mode of interaction of -BTX with AChR.
Dopamine Receptors: Signal Transduction in the D2 Receptor Family
Dopamine plays a central role in CNS neurotransmission, and its receptor systems have been associated with neuropathological disorders such as Parkinson's disease, schizophrenia, tardive dyskinesia and Huntington's chorea. Our main interest is to elucidate the molecular and functional basis of dopamine receptor diversity, and to understand how the various inhibitory D2 dopamine receptors (i.e. the D2, D3 and D4 receptors) orchestrate their manifold roles in the normal and diseased brain.
Although a high degree of homology exists between D2, D3 and D4, their coupling mechanisms appear to be distinct. We investigated the differential coupling of D2, D3 and D4 dopamine receptors to Gi, Gz and Gs in COS-7 cells that were transiently transfected with the individual murine dopamine receptors alone, as well as together with the a subunit of Gz. We demonstrated that the D2 dopamine receptor couples to Gi and to Gz to the same degree, whereas the D4 receptor couples more efficiently to Gz than to Gi. By contrast, D3 receptor can couple to Gz, and, albeit poorly, to Gi, but unlike D2 and D4, it also couples to the stimulatory G protein, Gs.
To further investigate the molecular basis for the differences between D2 and D3 dopamine receptors in their second messenger coupling, we have constructed and characterized four chimeras each composed of different segments of the original D2 and D3 receptors. We demonstrated that chimeras with a third cytoplasmic loop of the D2 receptor, couples to Gi protein, like D2 receptor. On the other hand chimeras containing a third cytoplasmic loop of the D3 receptor have coupling characteristic of the D2 receptor and couples also to Gs protein. These findings demonstrate that the third loop determines and accounts for the coupling of D2 and D3 dopamine receptors to G proteins.
Peripheral Markers for Schizophrenia
Through our research on dopamine receptors and acetylcholine receptors, we became interested in the possible association of these receptors with a number of neurological disorders, particularly schizophrenia. To date, a definitive diagnosis of schizophrenia is based on psychiatric and behavioral assessment and requires several months. There is neither an effective biological marker for identifying schizophrenia, nor a method for accurate and rapid diagnosis. Thus, there remains a vital need for a convenient assay for diagnosis and follow-up of schizophrenia. We attempt to develop reliable markers for schizophrenia by analyzing the correlation between schizophrenia and levels of dopamine receptors and of neuronal acetylcholine receptors in peripheral blood lymphocytes (PBLs). We have reported recently that there is a significant elevation of 2-6 folds in the levels of mRNA that encodes for the D3 dopamine receptor, in blood lymphocytes of schizophrenic patients, when compared with healthy controls.
In view of recent studies suggesting that neuronal 7 nicotinic acetylcholine receptor (7 AChR) may play a role in the pathogenesis of schizophrenia, we are also investigating now the levels of mRNA for 7 AChR in peripheral blood lymphocytes (PBLs), as another potential biological marker for schizophrenia. In a preliminary study we have observed a significant decrease (20-95%) of 7 mRNA levels in PBLs of schizophrenic patients, compared with controls. The decrease in 7 mRNA levels was not a result of medication and/or smoking. The availability of two different biological markers (D3 and 7) that can be both tested in PBLs makes the evaluation of schizophrenic patients by a peripheral and objective test, rather promising.
Dr. Revital Aricha, Postdoctoral Fellow
Dr. Revital Zahavi-Feferman, Research Associate
Dr. Sonia Berrih-Aknin, Visiting Scientist
Dr. Miriam C. Souroujon, Visiting Scientist
List of Publications (last 3 years)