Steven E. Pfeiffer

Does Paranode Formation and Maintenance Require Partitioning of Neurofascin 155 into Lipid Rafts

Paranodal axoglial junctions in myelinated nerve fibers are essential for efficient action potential conduction and ion channel clustering. We show here that, in the mature CNS, a fraction of the oligodendroglial 155 kDa isoform of neurofascin (NF-155), a major constituent of paranodal junctions, has key biochemical characteristics of a lipid raft-associated protein. However, despite its robust expression, NF-155 is detergent soluble before paranodes form and in purified oligodendrocyte cell cultures. Only during its progressive localization to paranodes is NF-155 (1) associated with detergent-insoluble complexes that float at increasingly lower densities of sucrose and (2) retained in situ after detergent treatment. Finally, mutant animals with disrupted paranodal junctions, including those lacking specific myelin lipids, have significantly reduced levels of raft-associated NF-155. Together, these results suggest that trans interactions between oligodendroglial NF-155 and axonal ligands result in cross-linking, stabilization, and formation of paranodal lipid raft assemblies.

Detergent-insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane

Glycosphingolipids and cholesterol form lateral assemblies, or lipid ‘rafts’, within biological membranes. Lipid rafts are routinely studied biochemically as low‐density, detergent‐insoluble complexes (in non‐ionic detergents at 4°C; DIGs, detergent‐insoluble glycosphingolipid/cholesterol microdomains). Recent discrepancies recommended a re‐evaluation of the conditions used for the biochemical analysis of lipid rafts. We have investigated the detergent insolubility of several known proteins present in the glycosphingolipid/cholesterol‐rich myelin membrane, using four detergents representing different chemical classes (TX‐100, CHAPS, Brij 96 and TX‐102), under four conditions: detergent extraction of myelin either at (i) 4°C or (ii) 37°C, or at 4°C after pre‐extraction with (iii) saponin or (iv) methyl‐β‐cyclodextrin (MβCD). Each detergent was different in its ability to solubilize myelin proteins and in the density of the DIGs produced. Brij 96 DIGs floated to a lower density than other detergents tested, possibly representing a subpopulation of DIGs in myelin. DIGs pre‐extracted with saponin were denser than DIGs pre‐extracted with MβCD. Furthermore, pre‐extraction with MβCD solubilized proteolipid protein (known to associate with cholesterol), whereas pre‐extraction with saponin did not, suggesting that saponin is less effective as a cholesterol‐perturbing agent than is MβCD. These results demonstrate that DIGs isolated by different detergents are not necessarily comparable, and that these detergent‐specific DIGs may represent distinct biochemical, and possibly physiological, entities based on the solubilities of specific lipids/proteins in each type of detergent.

Myelin glycosphingolipid/cholesterol-enriched microdomains selectively sequester the non-compact myelin proteins CNP and MOG.

Plasma membranes are complex arrays of protein and lipid subdomains. Detergent-insoluble, glycosphingolipid/cholesterol-enriched micro-domains (DIGCEMs) have been implicated in protein sorting and/or as sites for signaling cascades in the plasma membrane. We previously identified the presence of DIGCEMs in oligodendrocytes in culture and purified myelin and characterized a novel DIGCEM-associated tetraspan protein, MVP17/rMAL (Kim et al. (1995) Journal of Neuroscience Research 42, 413–422). We have now analyzed the association of known myelin proteins with DIGCEMs in order to provide a better understanding of their roles during myelin biogenesis. We used four well-established criteria to identify myelin DIGCEM-associated proteins: insolubility in a non-ionic detergent Triton X-100 at low temperature (4°C), flotation of the insoluble complexes to low density fractions in sucrose gradients, and TX-100 solubilization at 37°C, or at 4°C following treatment with the cholesterol-binding detergent saponin. We demonstrate that these proteins fall into four distinct groups. Although all tested proteins could be floated to a low-density fraction, proteolipid protein (PLP), myelin basic protein (MBP) and myelin associated glycoprotein (MAG) were solubilized by the detergent extraction, and connexin32 (Cx32) and oligodendrocyte-specific protein (OSP) met only some of the criteria for DIGCEMs. Only the non-compact myelin proteins 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) and myelin/oligodendrocyte glycoprotein (MOG) satisfied all four criteria for DIGCEM-associated proteins. Significantly, only ∼40% of CNP and MOG were selectively associated with DIGCEMs. This suggests that they may have both non-active “soluble”, and functionally active DIGCEM-associated, forms in the membrane, consistent with current views that DIGCEMs provide platforms for bringing together and activating components of the signal transduction apparatus. We therefore propose that CNP and MOG may have unique roles among the major myelin proteins in signaling pathways mediated by lipid-protein microdomains formed in myelin.

Human myelin proteome and comparative analysis with mouse myelin

Myelin, formed by oligodendrocytes (OLs) in the CNS, is critical for axonal functions, and its damage leads to debilitating neurological disorders such as multiple sclerosis. Understanding the molecular mechanisms of myelination and the pathogenesis of human myelin disease has been limited partly by the relative lack of identification and functional characterization of the repertoire of human myelin proteins. Here, we present a large-scale analysis of the myelin proteome, using the shotgun approach of 1-dimensional PAGE and liquid chromatography/tandem MS. Three hundred eight proteins were commonly identified from human and mouse myelin fractions. Comparative microarray analysis of human white and gray matter showed that transcripts of several of these were elevated in OL-rich white matter compared with gray matter, providing confidence in their detection in myelin. Comparison with other databases showed that 111 of the identified proteins/transcripts also were expressed in OLs, rather than in astrocytes or neurons. Comparison with 4 previous myelin proteomes further confirmed more than 50% of the identified proteins and revealed the presence of 163 additional proteins. A select group of identified proteins also were verified by immunoblotting. We classified the identified proteins into biological subgroups and discussed their relevance in myelin biogenesis and maintenance. Taken together, the study provides insights into the complexity of this metabolically active membrane and creates a valuable resource for future in-depth study of specific proteins in myelin with relevance to human demyelinating diseases.

Myelin‐Associated Galactolipids in Primary Cultures from Dissociated Fetal Rat Brain: Biosynthesis, Accumulation, and Cell Surface Expression

Abstract: Galactolipid metabolism was investigated as a function of development in primary cultures initiated from 19–21‐day‐old dissociated fetal rat brain. Significant amounts of galactocerebrosides, sulfatides, and monogalactosylglycerides were synthesized and accumulated by 8 days in culture. Thereafter the synthetic rates and levels of these galactolipids increased rapidly, reaching maximal values ∼22–29 days in culture. Galactolipids containing nonhydroxy or 2‐hydroxy fatty acid were both synthesized at approximately equal rates. The initial rates of synthesis, investigated at 15, 29, and 50 days in culture, were three‐ to fivefold higher for galactocerebrosides than for sulfatides and two‐ to threefold higher than for monogalactosylglycerides. The total number of cells staining with antisera against galactocerebroside of sulfatide also increased very rapidly between 8 and 22 days in culture, reaching levels of 4–5 million cells per seeded fetal brain. The amount of galactocerebroside or sulfatide per cell stained with the corresponding antiserum increased severalfold from 10 to 27 days in culture and remained high until at least 36 days in culture (the latest time point examined). Thus, the temporal expression of galactolipid accumulation in the cell cultures was comparable to that occurring in rat brain, but some important quantitative reductions in the levels of accumulation per cell in culture were noted. In addition, in contrast to normal brain in which galactolipid synthetic rates are reduced after the period of most active myelination, in culture both synthesis and turnover of these galactolipids remained high, suggestive of a partial arrest in myelin maturation.

The myelin–axolemmal complex: biochemical dissection and the role of galactosphingolipids

Myelin–axolemmal interactions regulate many cellular and molecular events, including gene expression, oligodendrocyte survival and ion channel clustering. Here we report the biochemical fractionation and enrichment of distinct subcellular domains from myelinated nerve fibers. Using antibodies against proteins found in compact myelin, non‐compact myelin and axolemma, we show that a rigorous procedure designed to purify myelin also results in the isolation of the myelin–axolemmal complex, a high‐affinity protein complex consisting of axonal and oligodendroglial components. Further, the isolation of distinct subcellular domains from galactolipid‐deficient mice with disrupted axoglial junctions is altered in a manner consistent with the delocalization of axolemmal proteins observed in these animals. These results suggest a paradigm for identification of proteins involved in neuroglial signaling.

Regulated Galactolipid Synthesis and Cell Surface Expression in Schwann Cell Line D6P2T

Clonal cell line D6P2T, subcloned from an ethylnitrosourea‐induced tumor line D6 of the rat peripheral nervous system, has been characterized with particular attention to galactolipid metabolism. Galactosylcerebroside and sulfatide synthesis and expression on the cell surface are highly regulated in D6P2T cells by mechanisms involving serum‐and cyclic AMP‐mediated pathways. These cells also express 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (Wolfgram protein Wla) and laminin. In contrast, myelin basic protein and antigen HNK‐1 were not detected. Line D6P2T appears to be a semi‐differentiated Schwann cell model, which offers interesting possibilities for studies of galactolipid synthesis, transport, and sorting.

Oligodendrocyte Development in Culture Systems

The oligodendrocyte has as its primary recognized function the task of producing a prodigious amount of membrane, possibly unequaled by any other cell type, and maintaining it for a lifetime. Morell and Norton (1980) estimate that during development, these otherwise rather diminutive cells produce several times their mass in myelin membrane each day. Additional interest is occasioned by the manner in which oligodendrocytes send multiple copies of this specialized membrane a substantial distance from their cell bodies before amplifying it into vast sheets that become wrapped and compacted around qualified axons (Peters, 1964; M. B. Bunge et al., 1962; Hirano, 1968; R. P. Bunge, 1968). Thus, oligodendrocytes engage in membrane biogenesis on a scale to intrigue the membrane biochemist.

Microglial cystatin F expression is a sensitive indicator for ongoing demyelination with concurrent remyelination

Demyelination coincides with numerous changes of gene expression in the central nervous system (CNS). Cystatin F, which is a papain‐like lysosomal cysteine proteinase inhibitor that is normally expressed by immune cells and not in the brain, is massively induced in the CNS during acute demyelination. We found that microglia, which are monocyte/macrophage‐lineage cells in the CNS, express cystatin F only during demyelination. By using several demyelinating animal models and the spinal cord tissues from multiple sclerosis (MS) patients, we examined spatiotemporal expression pattern of cystatin F by in situ hybridization and immunohistochemistry. We found that the timing of cystatin F induction matches with ongoing demyelination, and the places with cystatin F expression overlapped with the remyelinating area. Most interestingly, cystatin F induction ceased in chronic demyelination, in which remyelinating ability was lost. These findings demonstrate that the expression of cystatin F indicates the occurrence of ongoing demyelination/remyelination and the absence of cystatin F expression indicates the cessation of remyelination in the demyelinating area.

Myelin Biogenesis: Sorting out Protein Trafficking

Myelin biogenesis is a complex process involving coordinated exocytosis, endocytosis, mRNA transport and cytoskeletal dynamics. Recent studies indicate that soluble neuronal signals may control the surface expression of proteolipid protein, a process that involves reduced endocytosis and/or increased transport carrier recruitment from an intracellular pool.