PMCD

Figure 20 Evidence of lipid raft involvement in rHIgM22 signaling. (A-E) rHIgM22 immunostains patches of plasma membrane, as observed in serial z-sections throughout the cell (A—D). Stacking of the individual images shows the relative "patchiness" of the rHIgM22 staining pattern, suggesting that rHIgM22 antigen is localized to a specific plasma membrane microdomain. (F) rHIgM22 rescues premyelinating oligodendrocytes from death induced by treatment with hydrogen peroxide for 30 minutes. Cholesterol chelation with methyl-P-cyclodextrin (P-MCD) completely abrogates the protective effect of rHIgM22, suggesting that signaling from lipid rafts is critical for the ability of rHIgM22 to rescue oligodendrocytes from oxidative stress-induced apoptosis.

factor receptors closer to a threshold for full activation induced by substantially less extracellular growth factor. For example, PDGFa receptors (PDGFR) are sequestered in lipid rafts of oligodendrocytes committed to terminal differentiation, and PDGFR signaling from these rafts is required for survival of oligodendrocytes (Frost et al., 2003; Park et al., 2001). Likewise, axonal laminin-2 provides a target-dependent survival signal for differentiating oligodendro-cytes via signaling through the lipid raft localized a6pi integrin. Laminin-induced clustering of integrin within lipid rafts leads to substantial amplification of PI3-K and Akt signaling downstream from the PDGFR (Baron et al., 2003). Therefore, lipid raft microdomain-localized clustering of integrins and subsequent amplification of growth factor receptor signaling is an important signaling mechanism within oligodendrocytes. Similarly, integrin clustering within lipid rafts leads to a signaling cascade that uses Src and calcium calmodulin-dependent protein kinase II (CaMKII) activation to phosphorylate AMPA receptors, resulting in a slow-building potentiation of calcium flux through the AMPA receptor calcium channel (Kramar et al., 2003).

Clustering of oligodendrocyte cell surface proteoglycans within lipid raft microdomains initiates many of the same signaling events as integrin clustering (Simons and Horowitz, 2001). For example, syndecan-4 is a transmembrane heparan sulfate proteoglycan that is recruited to focal adhesions in response to extracellular matrix-dependent clustering or antibody-induced clustering (Lim et al., 2003; Tkachenko and Simons 2002). Syndecan-4 signals through protein kinase Ca (PKCa) by potentiating its response to phosphatidylinositol 4,5-bisphosphate (PIP2). This potentia-tion is accomplished by a clustering-dependent increase in the association of syndecan-4 with both PIP2 and PKCa,

Figure 2! A model of rHIgM22 and remyelination-promoting antibody signal induction. Pentameric rHIgM22 may bind to a surface antigen that is associated intracellularly with signal transduction elements that are activated by clustering. rHIgM22-mediated aggregation of cell surface epitopes translates into lipid raft clustering and concomitant induction of intracellular signals.

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Figure 2! A model of rHIgM22 and remyelination-promoting antibody signal induction. Pentameric rHIgM22 may bind to a surface antigen that is associated intracellularly with signal transduction elements that are activated by clustering. rHIgM22-mediated aggregation of cell surface epitopes translates into lipid raft clustering and concomitant induction of intracellular signals.

resulting in the formation of a multimeric ternary complex of these three molecules (Bass and Humphries, 2002; Lim et al., 2003). Syndecan-4 clustering also induces the activation of Rho family GTPases and FAK, leading to substantial reorganization of the actin cytoskeleton downstream from syndecan-4 multimerization (Yoneda and Couchman, 2003).

Proteoglycan clustering within lipid raft microdomains also results in substantial modification of calcium channel activity. NG2 is a transmembrane proteoglycan that is enriched on the surface of oligodendrocyte progenitors (Dawson et al., 2000). This proteoglycan associates with the glutamate receptor interaction protein (GRIP1) via a PDZ domain-mediated interaction (Stegmuller et al., 2003). As a result of this molecular linkage, NG2 is indirectly associated with the AMPA receptor within lipid rafts of oligodendro-cyte progenitors (Hering et al., 2003; Stegmuller et al., 2003). Immature oligodendrocyte AMPA channels are involved in regulation of proliferation and differentiation (Yuan et al., 1998), and the association of AMPA channels with NG2 suggests that adhesion-mediated clustering of NG2 may modify glial calcium signaling and thereby control maturation of oligodendrocytes. It is interesting to note that AMPA channel function is substantially modified by lectin binding, and concanavalin A binding leads to desensi-tization of the AMPA channel and a consequent increase in calcium permeability in the absence of changes in glutamate binding (Thalhammer et al., 2002). Moreover, sialidase treatment modulates AMPA function by alleviating desensi-tization, and this effect is indirect, as sialic acid residues are not found on the AMPA receptor (Hoffman et al., 1997). Because NG2 does bear sialic acid residues, it is reasonable to speculate that the effect of deglycosylation or lectin binding is mediated by an alteration in NG2 association with the AMPA receptor. Therefore, as with integrins, mul-timerization of proteoglycans is an important modulator of oligodendrocyte signaling, survival, proliferation, and differentiation.

The pentameric structure of the IgM antibodies we have identified as remyelination-promoting antibodies immediately suggests that clustering and multimerization of cell surface antigens is an important element in the signal trans-duction elicited downstream from antibody binding (Asakura et al., 1996, 1998; Bieber et al., 2001; Miller et al., 1994; Rodriguez and Lennon 1990; Sommer and Schachner 1981; Warrington et al., 2000). Therefore, we propose that our remyelination-promoting antibodies function as soluble initiators of the type of clustering described previously for integrins and proteoglycans. It is important to note that several of the antibodies we have shown to promote remyelina-tion are reactive with sialic acid residues (A2B5), other carbohydrate domains (HNK-1), and lipid raft-localized molecules (O1, O4) (Asakura et al., 1998; Warrington et al.,

2000). Moreover, several of these antibodies have been shown to alter oligodendrocyte morphology, initiate calcium signals, and induce differentiation (Dyer 1993; Dyer and Benjamins, 1989; 1990; 1991), and the general lack of similar effects induced by IgG antibodies reactive with similar epitopes suggests that multimerization is an important element in the transduction of these effects. It is also important to note, however, that cellular context is a critical factor in the signaling initiated by remyelination-promoting antibodies, as many of these antibodies recognize rather broad classes of molecules but only initiate signals in a limited manner. Thus we hypothesize that remyelination-promoting antibodies recognize oligodendrocyte surface molecules that are presented within specific contexts, and that this recognition event leads to the clustering of signal transducers within plasma membrane lipid raft microdomains.

Myelin reactive autoantibodies may also enhance repair through more indirect mechanisms. Antibodies binding to damaged oligodendrocytes and myelin may stimulate repair by enhancing the opsonization and clearance of myelin debris by macrophages (DeJong and Smith, 1997). Large numbers of macrophages are often observed in demyelinated lesions, and phagocytosis of myelin debris may be an important prerequisite to efficient remyelination. However, our most recent data indicate definitively that the Fc portion of the antibodies is not required for remyelination induced by sHIgM22 (Ciric et al., 2003), and this critical piece of information has focused our attention on the direct hypothesis.

Expanding the concept of antibody-mediated CNS repair from remyelination to axon regeneration, McKerracher and colleagues immunized animals with SCH before spinal cord hemisection or optic nerve crush (Ellezam et al., 2003; Huang et al., 1999). An identical preimmunization strategy was used, and therefore the antibody response is likely to be the same as in our earlier studies using the Theiler's virusmediated model of demyelination. Preimmunization with SCH resulted in significant axonal regrowth in both spinal cord and optic nerve lesion models and resulted in a functional recovery in the spinal cord lesion model. Sera from animals demonstrating axon regrowth contained increased titers of myelin-reactive antibodies, which, when used in vitro, allowed the outgrowth of neurites on normally inhibitory CNS myelin. It was hypothesized that anti-SCH antibodies promoted axon regeneration by blocking in vivo myelin-associated inhibitors of axon outgrowth (Ellezam et al., 2003; Huang et al., 1999). However, the SCH antisera did not contain elevated titers of antibodies to known myelin inhibitors (Nogo, myelin associated glycoprotein, chon-droitin sulfate proteoglycan). Therefore, myelin reactive antibodies might be useful not only to promote myelin repair after demyelinating disease but also for the treatment of axonal damage after spinal cord injury. Such antibodies may be administered exogenously or generated in vivo via appropriate immunization strategies. Recent work with the IN-1

antibody, also an oligodendrocyte reactive IgM, has led to very similar conclusions (Bregman et al., 1995; Chen et al., 2000; GrandPre et al., 2000).

There are currently few effective therapies to promote tissue repair or to prevent or reverse neurological deficits after CNS injury or disease; therefore, the characterization of endogenous immune-mediated repair mechanisms is important. An understanding of these mechanisms should open up significant new areas for the development of antibody-based therapeutics and perhaps small molecule-based therapeutics and vaccines for induction of the reparative response. Remyelination is an important therapeutic goal for the treatment of neurological disease and is likely to protect axons from further injury. Human monoclonal antibodies that promote remyelination represent a new class of therapeutics for diseases such as MS, spinal cord injury, neurodegeneration, and stroke.

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