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Conference
on Myelin Structure and Its Role in Autoimmunity II
European Science Foundation. MARIE Network May 1-5, 2005. Potenza, Basilicata, Italy. I. Overview of MARIE Network and ESF Conference Three years ago, an ESF Exploratory Workshop and ISN Small Conference on MYELIN STRUCTURE AND ITS ROLE IN AUTOIMMUNITY was held in Potenza, Basilicata, Italy, from 5-8 June 2002. The conference was immensely successful, with over 70 scientists participating from across Europe and from around the globe. The overwhelming consensus amongst the attendees was that it is important to decipher how myelin is made and structured in order to understand the mechanisms of assembly, function, and disruption in its physiopathology. On these grounds, an ESF Network called MARIE (Myelin Autoimmunity Research in Europe) was established with a 3-year mandate (January 2004 - December 2006) to promote interdisciplinary collaboration in this field. The activities of the MARIE Network are coordinated by a Steering Committee with several International Observers. Further information on the Network can be found at the website http://www.marienetwork.org/. Under the auspices
of the MARIE network and as an integral part of its activities, the ESF
Conference on "Myelin Structure and its Role in Autoimmunity II"
was held again at the Giubileo Hotel, near Potenza, Italy. The conference
center was very pleasant, and all participants were comfortably accommodated
on-site, thereby facilitating a good deal of interaction. In total, 64
registered participants attended the meeting: 49 were from Europe (21
from Italy, 8 from Germany, 4 each from Sweden, Switzerland, the United
Kingdom, 3 from France, 2 from Austria, 2 from Belgium, and 1 from The
Netherlands. There were also 4 participants from Canada, 7 from the United
States, 2 from Australia, and 1 each from Japan and Singapore. A total
of 24 young scientists (doctoral students and postdoctoral researchers)
attended. Of the total participants, 26 were female. II. Themes of the ESF Conference After opening remarks by Professors Paolo Riccio (Chair of MARIE Steering Committee) and George Harauz (International Observer), the remainder of the meeting was devoted to scientific sessions, organized into three themes, with specific questions discussed at the end of the day in a round table format: 1 - The myelin sheath and its proteins What is changing in
our knowledge of myelin structure? 2 - Generating and disrupting myelin What is the role of
metalloproteinases in myelin breakdown and regeneration? 3 - Autoimmunity, molecular mimicry and therapy How do lipid rafts
relate to myelin compaction and signaling? The format of the
conference comprised: The key questions listed above were presented simply as starting points, but the discussions evolved naturally into many directions, and were lively and stimulating. III. Scientific Sessions of the ESF Conference A summary of the topics
of the scientific presentations follows, showing a healthy diversity,
and a synergy that was created and facilitated by this meeting. Chair: Jan Sedzik, Robert Zand Myelin structure and
stability in murine transgenics (Dan Kirschner, Boston, MA, USA) Session summary (Jan
Sedzik and Robert Zand) - Chair: Dan Kirschner, Eugenia Polverini MBP: how conformational
adaptibility generates functional diversity (George Harauz, Guelph, ON,
Canada) Session summary (George
Harauz) - Myelin basic protein (MBP) is a family of developmentally-regulated
proteins that arise from different transcription start sites of the Golli
(genes of the oligodendrocyte lineage) gene complex, and by alternate
splicing to generate different isoforms. The best-studied member of this
family is the 18.5 kDa "classic" MBP, that is further diversified
by a plethora of post-translational modifications. These changes result
in the cumulative reduction of MBP's net positive charge, and affect its
interactions with membranes and with other proteins. Structurally, MBP
is a highly-flexible and extended molecule of the emerging "intrinsically-unstructured"
or "conformationally adaptable" class that is now known to comprise
several hundred proteins, including many involved in signalling. It has
been suggested that MBP functions in signal transduction mechanisms involved
in cytoskeletal organisation, in addition to its primary role as a membrane
adhesion molecule. The plenary lectures described studies of MBP structure,
and its interactions, using a wide variety of biochemical, biophysical,
and computational tools. We do not yet know MBP's 3D structure, but NMR
spectroscopy promises to yield information in this regard within the next
several years. The problem is difficult because MBP's conformation depends
strongly on its environment, including its association with other molecules,
and on other factors such as surface pressure. We also learned that modifications
to MBP modulate its interactions with lipids and other proteins, and effect
its partitioning into membrane microdomains in myelin. At the molecular
level, then, better biochemical and proteomic dissection tools are required
to establish precisely how MBP is partitioned in various myelin subdomains.
In contrast, other myelin proteins such as P2 have been well-studied by
X-ray crystallography, whereas proteolipid protein (PLP) is so refractory
to experimental manipulation that most of our knowledge about it is based
on studies of selected domains. Chair: Kazuhiro Ikenaka, Klaus-Armin Nave Cholesterol in myelination
(Gesine Saher and Klaus-Armin Nave, Goettingen, Germany) Chair: Ghislain Opdenakker, Claude Bernard Role of proteolytic
enzymes in myelin turnover: a balance between deleterious versus protective
events in EAE and MS (Ghislain Opdenakker, Leuven, Belgium) Session summary (Ghislain
Opdenakker and Claude Bernard) - This session was introduced by an overview
of the roles played by extracellular enzymes in demyelinating diseases,
their regulation by gene transcription (induced by cytokines and chemokines),
by chemokine-mediated secretion, by activation, inhibition and glycosylation.
Serine proteases, such as plasminogen activators and plasmin, and matrix
metalloproteinases (MMPs), such as gelatinase B/ MMP-9, not only form
an enzyme cascade that mediates the breakdown of basement membranes in
the central and peripheral nervous system, but these enzymes also cleave
protein substrates into peptides before these are entered into the intracellular
pathways of antigen processing and MHC-presentation to T-cells. For instance,
myelin basic protein, but alsoaB-crystallin, two notable auto-antigens
in multiple sclerosis, are cleaved by gelatinase B/ MMP-9 in such a way
that immunogenic peptides are produced as "remnant epitopes generating
autoimmunity" (REGA-model), before these are intracellularly or extracellularly
loaded into MHC proteins. These findings have therapeutical implications
in that targeting specific proteases may have beneficial effects, as is
already demonstrated with MMP-9 knockout mice and D-penicillamine and
tetracyclines as MMP inhibitors. Chair: Claude Bernard, Hartmut Wekerle The autoimmune pathogenesis
of MS (Hartmut Wekerle, Martinsried bei Muenchen, Germany) Chair: Chris Linington, Claude Genain Autoantibodies and
MOG in the pathogenesis of MS (Chris Linington, Aberdeen, UK) Session summary (Chris
Linington) - Autoantibody responses directed against the extracellular
domain of the myelin oligodendrocyte glycoprotein (MOG) play a crucial
role in the pathogenesis of immune-mediated demyelination in experimental
autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis.
This session discussed the exciting advances made over the past two years
in defining the structure of MOG, identifying epitopes recognised by demyelinating
autoantibodies and investigating the pathophysiological relevance of autoimmune
responses to this antigen in human disease. Constanze Breithaupt (Germany)
presented her data on the crystal structure of the extracellular domain
of rat MOG complexed to a humanized Fab fragment of a demyelinating MOG-specific
monoclonal antibody, 8-18C5. This antibody recognizes an immunodominant
epitope targeted by demyelinating autoantibodies in H2-d mice. The availability
of the three-dimensional complex formed between MOG and the Fab domain
of the antibody represents a major advance in our understanding of the
immunobiology of antibody-mediated demyelination and is one of only three
complex structures involving autoantigens that have been determined so
far. The extracellular domain of MOG forms a compact but otherwise classical
Ig V-like fold the upper surface of which is recognized by the antibody.
The epitope is highly discontinuous although the FG loop (amino acids
101-108) contributes >65% to the total contact surface. Mutagenesis
studies demonstrated the dominance of this structure in determining the
epitope specificity of the antibody response to MOG as exchanging only
two amino acids within the FG-loop eliminated the binding of 9 out of
10 MOG-specific monoclonal antibodies. A different approach to map out
epitope complexity was presented by Claude Genain (U.S.A.) whose group
has generated recombinant MOG-specific antibodies derived from a marmoset
sensitized with the rat autoantigen. Competition studies using these reagents
suggest that in the marmoset the antibody response to MOG is also restricted
to discontinuous epitopes but that these are distinct from those recognized
by 8-18C5. However, as the recombinant marmoset antibodies inhibit the
recognition of MOG by antibodies in MS sera it is probable that they recognize
clinically relevant epitopes. The functional/clinical relevance of the
anti-MOG response was addressed by several presenters, in particular Andreas
Lutterotti (Austria) who discussed the diagnostic/prognostic relevance
of MOG-specific antibodies in human disease. There was agreement that
maintaining the correct conformation of the extracellular domain of MOG
is essential if an assay is to detect pathologically relevant autoantibodies,
although responses to linear epitopes that become accessible when the
protein is denatured may provide useful prognostic data. However, the
clinical significance of MOG as a target for antibody mediated demyelination
remains to be formally demonstrated and. as discussed by both Claude Genain
and Emily Mathey (U.K.), there are many potential targets for antibody-mediated
pathomechanisms in the pathological CNS. In this context Genain presented
data indicating that galactosyl ceramide-specific antibodies may be involved
in disease pathogenesis in certain patients with MS, while Mathey discussed
the potential role of autoantibody responses directed against the oligodendrocyte
progenitors. In the latter case it was demonstrated that autoimmune responses
directed against the NG2 proteoglycan, a classical cell surface marker
for oligodendrocyte progenitors, dramatically enhances disease severity
in MOG-induced EAE. NG2-specific antibodies have been identified in the
csf of patients with MS suggesting that antibody responses to this and
other progenitor cell autoantigens could play an unexpected role in the
pathogenesis of MS. Previously it was assumed that such antibody mediated
effects were due to the activation of immune effector mechanisms such
as the complement cascade, however the final presentation by Cecilia Marta
(U.S.A.) questioned this somewhat entrenched view. Previous studies demonstrated
that 8-18C5 binding to the Ig domain of MOG leads to the depolymerisation
of oligodendrocyte microtubuli in vitro. Dr Marta demonstrated that not
only is this associated with the segregation of MOG into lipid rafts,
but also even in the absence of cross-linking this results in protein
phosphorylation indicative of intra-cellular signalling. This observation
may result in a dramatic reassessment of how autoantibodies influence
disease activity in MS, rather than dramatic immune mediated events leading
to gross-tissue destruction, the low titres of antibody present in most
patients may mediate functional effects that compromise the long term
stability of the myelin sheath or axon. Chair: Sergio Baranzini, Robert Weissert, Gabriella Coniglio Molecular dynamics
of demyelination: functional genomics in EAE (Sergio Baranzini, San Francisco,
CA, USA) Chair: Massimo Degano, Marie-Paule Lefranc Crystal structure of CD1d bound to phosphatidylcholine: a molecular basis for the activation of regulatory NKT cells (Massimo Degano, Milano, Italy) Informatics technologies
for turning data into biological knowledge (Vladimir Brusic, Singapore) Session summary (Massimo Degano) - Despite the advancements in the fields of medicine, biochemistry, and immunology, the etiopathogenesis of multiple sclerosis, and of several other demyelinating diseases, remains unclear. Nevertheless, it is well appreciated that the autoimmune component plays a central role in mediating the damage to the myelin sheaths. Deciphering the nature and role of the individual components that are involved in the autoimmune response to myelin antigens is currently one of the most challenging goals in MS research. Moreover, it is becoming increasingly clear that it is crucial to combine together the wealth of data derived from classic, reductionist approaches using complex algorithms to extrapolate functional data. The purpose of this session, chaired by Massimo Degano and Marie-Paule Lefranc, was to present new approaches and results in structural studies aimed at therapeutic approaches in disease. Massimo Degano introduced the session with a brief overview of the results recently published by Hahn et al. (Nature Immunology, May 2005), who determined the structure of the ternary complex between the MHC class II (HLA-DR2) molecule bound to the MBP85-99 peptide and a MS-associated T cell receptor (TCR). The study showed a different mode of TCR interaction with the MHC-peptide complex, compared to anti-viral or anti-bacterial receptors, and suggests how autoreactive TCR could escape negative selection in the thymus. Massimo Degano then presented the crystal structure of the mouse CD1d molecule bound to a self phospholipid. The structure offers novel insights in the molecular mechanism that activates regulatory NKT cells, crucial for the maintenance of peripheral tolerance. Vladimir Brusic underscored the importance of updating and filtering the existing information in databases, and showed a real-life example in the study of allergens. Marie-Paule Lefranc presented the status and recent updates of the IMGT web site, that now extends to the analysis of TCRs and MHC molecules. The database is a crucial reference point for analysis of immunoglobulin and MHC superfamilies, including their binding specificities. Finally, Nikolai Petrovsky showed how mathematical models can be applied to immunological studies to predict the outcome of disease. The active discussion at the end of the presentations confirmed that the community increasingly appreciates the knowledge derived from these "crossover" approaches, who represent a new frontier for autoimmunity studies. IV. Oral presentations of young investigators. An important outcome of the meeting was the participation of postgraduate students as well as young scientists, all of whom presented posters. Their exposure to good "frontier" science, as well as the possibility for them to meet more senior colleagues and discuss with them their research endeavours, was for many a unique opportunity and a most valuable experience. Several of the young investigators also presented their posters orally: Solution NMR structure of an immunodominant epitope of MBP (Christophe Fares, Goettingen, Germany); Native myelin oligodendrocyte glycoprotein (MOG) is critical for severe chronic neurological disease and demyelination (Paul Smith, Rijswijk, The Netherlands); Antibodies against native MOG in patients with MS (Susanne Gaertner, Tuebingen, Germany); Dynamics of T lymphocyte activation in adoptive transfer EAE (Kieran Williams, Aberdeen, United Kingdom); Characterization of class-I MHC restricted peptides of myelin in MS patients (Laureline Berthelot, Nantes, France); Epitope specificity of serum antibodies against the extracellular domain of myelin oligodendrocyte glycoprotein (MOG-Ig) during immunomodulatory therapies in MS patients (Michael Kahlil, Innsbruck, Austria). Poster presentations alone were as follows: Acid sphingomyelinase knockout mice: PNS alterations (Alessandra Gilardini, Monza, Italy); Partitioning of proteins in PNS myelin microdomains (Giulia Carlone, Potenza, Italy); The pH dependent unfolding mechanism of P2 myelin protein: an experimental and computational study (Eugenia Polverini, Parma, Italy); Molecular motions in oriented lipid bilayers containing myelin basic protein: an incoherent neutron scattering study (Ranieri Rolandi, Genova, Italy). ---------------------------------------------------------------------------------------------------------- Summing-up of the
MARIE Conference by George Harauz, Ghislain Opdenakker, Chris Linington,
Vladimir Brusic and Paolo Riccio. V. Assessment of Results - Outcome and future contributions to the field The novel formula that the organizers had adopted for the meeting was highly successful in that it allowed strong and fruitful interactions amongst workers in various disciplines, namely, biophysics, cell and molecular biology, neuroimmunology, bioinformatics and clinical neurology. Platform presentations were balanced as much as possible to provide equal exposure to all disciplines, and the relationships and complementation amongst different fields were promoted. The scientific topics were well chosen in definite fields, and were organized in such way that structure was the first and the ultimate goal of a cycle of reports, starting from the myelin membrane and arriving to the peptides presented by the antigen presenting cells. Complex diseases such as multiple sclerosis require complex strategies to overcome them. Complex is seen here as multifactorial. In other words, a multifactorial disease such as MS requires a multidisciplinary approach and, in turn, the capability of expand our own specific knowledge to other fields, to be imaginative and see new aspects, and to recognize the complex problem in its intrinsic simplicity, and solve it. Thus, the integration of different types of knowledge and expertise in different fields was intended not only to generate a common language, but also to formulate a more general vision of the problem. It is expected that this conference will spread the seeds for future collaborations amongst individuals in different fields. A final initiative that was discussed was the compilation of a book, devoted to techniques in myelin research, with chapters written by the participants. Such a monograph would have the effects of disseminating the scientific activities of the MARIE network more widely, and would also be of educational value to newcomers to the field. Usage of the ISN money
for MARIE Conference: Paolo Riccio, Potenza
and Bari, Italy (riccio@unibas.it) |
