Experimental autoimmune encephalomyelitis in the rat.

There are several diverse rat models of experimental autoimmune encephalomyelitis (EAE) that can be used to investigate the pathogenesis and regulation of autoimmunity against CNS myelin. The disease course of these models ranges from an acute monophasic disease with limited demyelination to a chronic relapsing or chronic progressive course marked by severe demyelination. These models enable the study of encephalitogenic T cells and demyelinating antibody specific for major neuroantigens such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or proteolipid protein (PLP), among other important CNS autoantigens. Overall, this unit provides an overview of common methods for induction of active and passive EAE, assessment and analysis of clinical disease, preparation and purification of myelin basic protein, and derivation of neuroantigen-specific rat T cell lines. This unit also provides a brief discussion of the basic characteristics of these models.

Sonic hedgehog-dependent emergence of oligodendrocytes in the telencephalon: evidence for a source of oligodendrocytes in the olfactory bulb that is independent of PDGFRalpha signaling.

Most studies on the origin of oligodendrocyte lineage have been performed in the spinal cord. By contrast, molecular mechanisms that regulate the appearance of the oligodendroglial lineage in the brain have not yet attracted much attention. We provide evidence for three distinct sources of oligodendrocytes in the mouse telencephalon. In addition to two subpallial ventricular foci, the anterior entopeduncular area and the medial ganglionic eminence, the rostral telencephalon also gives rise to oligodendrocytes. We show that oligodendrocytes in the olfactory bulb are generated within the rostral pallium from ventricular progenitors characterized by the expression of PLP: We provide evidence that these Plp oligodendrocyte progenitors do not depend on signal transduction mediated by platelet-derived growth factor receptors (PDGFRs), and therefore propose that they belong to a different lineage than the PDGFRalpha-expressing progenitors. Moreover, induction of oligodendrocytes in the telencephalon is dependent on sonic hedgehog signaling, as in the spinal cord. In all these telencephalic ventricular territories, oligodendrocyte progenitors were detected at about the same developmental stage as in the spinal cord. However, both in vivo and in vitro, the differentiation into O4-positive pre-oligodendrocytes was postponed by 4-5 days in the telencephalon in comparison with the spinal cord. This delay between determination and differentiation appears to be intrinsic to telencephalic oligodendrocytes, as it was not shortened by diffusible or cell-cell contact factors present in the spinal cord.

Production of a recombinant hybrid molecule of cholera toxin-B-subunit and proteolipid-protein-peptide for the treatment of experimental encephalomyelitis.

Mucosal administration of experimental autoimmune encephalomyelitis (EAE)-specific autoantigens can reduce the onset of disease. To examine whether cholera toxin-B-subunit (CTB)-conjugated EAE-specific T-cell epitope can reduce development of the autoimmune disease in mice, we produced a recombinant hybrid molecule of CTB fusion protein linked with proteolipid-protein (PLP)-peptide139-151(C140S) at levels up to 0.1 gram per liter culture media in Bacillus brevis as a secretion-expression system. Amino acid sequencing and GM1-receptor binding assay showed that this expression system produced a uniformed recombinant hybrid protein. EAE was induced in SJL/J mice by systemic administration with the PLP-peptide. When nasally immunized 5 times with 70 microg rCTB PLP-peptide hybrid protein, mice showed a significantly suppressed development of ongoing EAE and an inhibition of both the PLP-peptide-specific delayed-type hypersensitivity (DTH) responses and leukocyte infiltration into the spinal cord. In contrast, all mice given the PLP-peptide alone or the PLP-peptide with the free form of CTB did not suppress the development of EAE and DTH responses. These results suggest that nasal treatment with the recombinant B. brevis-derived hybrid protein of CTB and autoantigen peptide could prove useful in the control of multiple sclerosis.

Quantification of self-recognition in multiple sclerosis by single-cell analysis of cytokine production.

Identifying and quantifying autoaggressive responses in multiple sclerosis (MS) has been difficult in the past due to the low frequency of autoantigen-specific T cells, the high number of putative determinants on the autoantigens, and the different cytokine signatures of the autoreactive T cells. We used single-cell resolution enzyme-linked immunospot (ELISPOT) assays to study, directly ex vivo, proteolipid protein (PLP)-specific memory cell reactivity from MS patients and controls. Overlapping 9-aa-long peptides, spanning the entire PLP molecule in single amino acid steps, were used to determine the frequency and fine specificity of PLP-specific lymphocytes as measured by their IFN-gamma and IL-5 production. MS patients (n = 22) responded to 4 times as many PLP peptides as did healthy controls (n = 22). The epitopes recognized in individual patients, up to 22 peptides, were scattered throughout the PLP molecule, showing considerable heterogeneity among MS patients. Frequency measurements showed that the number of PLP peptide-specific IFN-gamma-producing cells averaged 11 times higher in MS patients than in controls. PLP peptide-induced IL-5-producing T cells occurred in very low frequencies in both MS patients and controls. This first comprehensive assessment of the anti-PLP-Th1/Th2 response in MS shows a greatly increased Th1 effector cell mass in MS patients. Moreover, the highly IFN-gamma-polarized, IL-5-negative cytokine profile of the PLP-reactive T cells suggests that these cells are committed Th1 cells. The essential absence of uncommitted Th0 cells producing both cytokines may explain why therapeutic strategies that aim at the induction of immune deviation show little efficacy in the established disease.

Effect of ATP depletion on the palmitoylation of myelin proteolipid protein in young and adult rats.

The present study was designed to determine whether the palmitoylation of the hydrophobic myelin proteolipid protein (PLP) is dependent on cellular energy. To this end, brain slices from 20- and 60-day-old rats were incubated with [3H]palmitate for 1 h in the presence or absence of various metabolic poisons. In adult rats, the inhibition of mitochondrial ATP production with KCN (5 mM), oligomycin (10 microM), or rotenone (10 microM) reduced the incorporation of [3H]palmitate into fatty acyl-CoA and glycerolipids by 50-60%, whereas the labeling of PLP was unaltered. Incubation in the presence of rotenone (10 microM) plus NaF (5 mM) abolished the synthesis of acyl-CoA and lipid palmitoylation, but the incorporation of [3H]palmitate into PLP was still not different from that in controls. In rapidly myelinating animals, the inhibition of both mitochondrial electron transport and glycolysis obliterated the palmitoylation of lipids but reduced that of PLP by only 40%. PLP acylation was reduced to a similar extent when slices were incubated for up to 3 h, indicating that exogenously added palmitate is incorporated into PLP by ATP-dependent and ATP-independent mechanisms. Determination of the number of PLP molecules modified by each of these reactions during development suggests that the ATP-dependent process is important during the formation and/or compaction of the myelin sheath, whereas the ATP-independent mechanism is likely to play a role in myelin maintenance, perhaps by participating in the periodic repair of thioester linkages between the fatty acids and the protein.

Overproduction and immuno-affinity purification of myelin proteolipid protein (PLP), an inositol hexakisphosphate-binding protein, in a baculovirus expression system.

Myelin proteolipid protein (PLP) is a major integral membrane protein of central nervous system myelin and is considered to play a significant role in myelination. PLP has a four-transmembrane structure, judging from the hydropathy profile. In addition, it has InsP6 binding activity. Here, we have succeeded in producing PLP in large quantities of 3.9 pg/cell (6 mg/L) by using a baculovirus expression system and developing an efficient purification method, maintaining InsP6 binding activity. The recombinant PLP (rPLP) was purified by ion-exchange and immunoaffinity chromatography in a nonorganic solvent. The final yield of purified rPLP was 36%. The Kd and Bmax values for the InsP6-PLP binding were 55 nM and 33 pmol/microgram protein, respectively. The Kd value of purified rPLP is equal to that of mouse brain PLP. These results indicate that purified rPLP keeps its native conformation and binds InsP6 in an almost one-to-one ratio.

Myelin proteolipid protein (PLP), but not DM-20, is an inositol hexakisphosphate-binding protein.

Myelin proteolipid protein (PLP) and its alternatively spliced isoform, DM-20, are the major integral membrane proteins of central nervous system myelin. It is known that PLP and DM-20 are delivered to myelin by a finely regulated vesicular transport system in oligodendrocytes. Evolutionarily, it is believed that ancestral DM-20 acquired a PLP-specific exon to create PLP, after which PLP/DM-20 became a major component of central nervous system myelin. We purified PLP as an inositol 1,3,4,5-tetrakisphosphate-binding protein after solubilization in a non-organic solvent. However, under the isotonic condition, PLP binds inositol hexakisphosphate (InsP6) significantly, not inositol 1,3,4,5-tetrakisphosphate. Most of the InsP6-binding proteins are involved in vesicular transport, suggesting the involvement of PLP in vesicular transport. We separated DM-20 from PLP by CM-52 chromatography and showed that DM-20 has no InsP6 binding activity. These findings indicate that the PLP-specific domain confers the InsP6 binding activity and this interaction may be important for directing PLP transport to central nervous system myelin.

Preparation of giant myelin vesicles and proteoliposomes to register ionic channels.

Myelin vesicles, reconstituted liposomes with proteolipid protein (PLP), the main protein component of myelin, and electrophysiological patch-clamp are potentially powerful tools to study the role of myelin in functional ionic channels. However, technical difficulties in the vesiculation of myelin and the small size of the vesicles obtained do not permit the application of micropipettes for current recordings. From a suspension of purified myelin we have prepared oligolamellar vesicles (mean diameter of 144 nm) using the so-called French pressure system. From this preparation we obtained giant myelin vesicles approximately 10 microns in mean diameter, using a dehydration-rehydration procedure. Qualitative analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no significant loss of any component in these vesicles due to pressure, in comparison with non-vesiculated myelin. A way of preparing giant liposomes of approximately 80-100 microns and proteoliposomes of approximately 30 microns in mean diameter, using the same dehydration-rehydration procedure, is also reported. Reconstitution of purified PLP in giant liposomes was confirmed by fluorescent labeling of PLP and by fluorescence microscopy. The current recordings from these vesicles prove the validity of these methods and provide significant evidence of the existence of ionic channels in myelin membranes and the possibility that PLP functions as a channel. The physiological significance and characterization of these channels remain yet unresolved. These results have a special significance for elucidating the molecular role of myelin in the regulation of neural activity and in the brain ion microenvironment.