Multiple Splice Isoforms of Proteolipid M6B in Neurons and Oligodendrocytes

Proteolipids are abundant integral membrane proteins, initially described as structural proteins of CNS myelin. More recently, two neuronal proteins related to proteolipid protein (PLP), termed M6A and M6B, were identified, suggesting a common function of proteolipids in oligodendrocytes and neurons. We have analyzed the X-linked M6B gene and discovered an unexpected complexity of protein isoforms. Two promoters and alternative exons yield at least eight M6B proteins and polypeptides, differentially expressed in neurons and oligodendrocytes. Six isoforms are tetraspan membrane proteins that differ by highly conserved amino- and carboxy-terminal domains, termed alpha, beta, psi, and omega. In MDCK cells, the beta-domain of M6B stabilizes tetraspan proteolipids at the cell surface, whereas non-beta isoforms are more abundant in intracellular compartments. Cotransfection experiments suggest a physical interaction of M6B and mutant PLP, when retained in the endoplasmic reticulum, that may also contribute to oligodendrocyte dysfunction in Pelizaeus-Merzbacher disease.     

Classic and soma-restricted proteolipids are targeted to different subcellular compartments in oligodendrocytes

The myelin proteolipid (PLP) gene is very active in oligodendrocytes (OLs) and generates at least four proteins: the classic PLP and DM20 proteolipids, which are associated with compact myelin and the srPLP and srDM20, which are associated with the cell soma. These proteins are extremely hydrophobic and appear to follow the biosynthetic route used by secretory proteins. In this study, we have analyzed the subcellular distribution of the newly described sr-proteolipids and compared it to that of the classic proteolipids. Immunocytochemical analysis indicates that the sr-proteolipids and classic proteolipids are found in association with the endoplasmic reticulum (ER) and Golgi apparatus of mature OLs in vitro. Whereas the classic proteolipids become associated with the myelin-like sheets elaborated by OLs, the sr-proteolipids are not targeted to the myelin leaflets. The sr-proteolipids were associated with endosomes and with recycling vesicles as determined by double immunocytochemistry with markers such as syntaxin 6 and clathrin. In vivo, immunohistochemical analysis showed a distribution of the sr-proteolipids that was similar to that obtained in vitro, with a total absence of incorporation of sr-proteolipids into compact myelin. This differential subcellular localization is further evidence for a biological role for these products of the PLP/DM20 gene, which is different from that of the classic proteolipids.     

His36Pro point-mutated proteolipid protein retained in the endoplasmic reticulum of oligodendrocytes in the shaking pup

The shaking pup (shp) is a canine mutation that affects the myelin protein proteolipid protein (PLP) and its smaller and less abundant isoform, DM20, with proline replacing histidine(36), resulting in a severe myelin deficiency in the central nervous system. We present evidence that the mutation leads to disrupted trafficking of the shp PLP/DM20 within oligodendrocytes. Immunohistochemical studies revealed significantly reduced levels of PLP/DM20 and other major myelin components such as myelin basic protein (MBP), myelin associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in shp myelin. The distribution of shp PLP/DM20 proteins were altered and mostly retained in perinuclear cytoplasm and proximal processes, which co-localized with distended rough endoplasmic reticulum (RER) within oligodendrocytes. No abnormal accumulation of MAG, MBP, or CNP in the cell body was found. These results suggest that mutated PLP/DM20 in the shp could be selectively retained in RER, causing disruption of their translocation to the periphery to myelinate axons.     

Early ultrastructural defects of axons and axon–glia junctions in mice lacking expression of Cnp1

Most axons in the central nervous system (CNS) are surrounded by a multilayered myelin sheath that promotes fast, saltatory conduction of electrical impulses. By insulating the axon, myelin also shields the axoplasm from the extracellular milieu. In the CNS, oligodendrocytes provide support for the long‐term maintenance of myelinated axons, independent of the myelin sheath. Here, we use electron microscopy and morphometric analyses to examine the evolution of axonal and oligodendroglial changes in mice deficient in 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) and in mice deficient in both CNP and proteolipid protein (PLP/DM20). We show that CNP is necessary for the formation of a normal inner tongue process of oligodendrocytes that myelinate small diameter axons. We also show that axonal degeneration in Cnp1 null mice is present very early in postnatal life. Importantly, compact myelin formed by transplanted Cnp1 null oligodendrocytes induces the same degenerative changes in shiverer axons that normally are dysmyelinated but structurally intact. Mice deficient in both CNP and PLP develop a more severe axonal phenotype than either single mutant, indicating that the two oligodendroglial proteins serve distinct functions in supporting the myelinated axon. These observations support a model in which the trophic functions of oligodendrocytes serve to offset the physical shielding of axons by myelin membranes. © 2009 Wiley‐Liss, Inc.     

Proteolipid protein interactions in transfectants: Implications for myelin assembly

The proteolipid proteins (PLP and DM20) are major constituents of CNS myelin, but how they are delivered to and organized within the oligodendrocyte plasma membrane is incompletely understood. We have expressed both PLP and DM20 singly or together in a host cell line, HeLa. In either DM20 or PLP transfectants, at early time points (24 hours), the expressed proteins are found within intracellular compartments. In DM20 transfectants, the protein is delivered to the plasma membrane by 48 hours. In HeLa cells, PLP remains intracellular when expressed in the absence of DM20; only when it is coexpressed with DM20 is it transported to the plasma membrane. In cotransfectants, PLP can also be localized to organelles involved in both the protein biosynthetic and the endocytic pathways. Since, in HeLa cells at least, the delivery of PLP to the plasma membrane is facilitated by the coexpression of DM20, we suggest that the two proteins interact intracellularly to form a complex. In some PLP/DM20 cotransfectants, the proteolipids are concentrated in regions of cell-cell contact. The regional accumulation of these proteins at cell-cell interfaces is highly reminiscent of the behavior in transfected cells of another myelin protein, P_o, and certain cadherin polypeptides, both of which have readily demonstrable membrane adhesive properties. Our data suggests that at certain stoichiometric ratios, proteolipids can become stablized at cell surfaces to form adhesive bonds. © 1994 Wiley-Liss, Inc.     

PLP/DM20 Expression and turnover in a transgenic mouse model of pelizaeus‐merzbacher disease

The most common cause of Pelizaeus‐Merzbacher (PMD) is due to duplication of the PLP1 gene but it is unclear how increased gene dosage affects PLP turnover and causes dysmyelination. We have studied the dynamics of PLP/DM20 in a transgenic mouse model of PMD with increased gene dosage of the proteolipid protein gene (Plp1). The turnover of PLP/DM20 were investigated using an ex‐vivo brain slice system and cultured oligodendrocytes. Homozygous mice have reduced PLP translation, markedly enhanced PLP degradation, and markedly reduced incorporation of PLP into myelin. Proteasome inhibition (MG132) prevented the enhanced degradation. Numerous autophagic vesicles are present in homozygous transgenic mice that may influence protein dynamics. Surprisingly, promoting autophagy with rapamycin decreases the degradation of nascent PLP suggesting autophagic vacuoles serve as a cellular storage compartment. We suggest that there are multiple subcellular fates of PLP/DM20 when overexpressed: the vast majority being degraded by the proteasome, a proportion sequestered into autophagic vacuoles, probably fused with endolysosomes, and only a small proportion entering the myelin sheath, where its association with lipid rafts is perturbed. Transgenic oligodendrocytes have fewer membrane sheets and this phenotype is improved with siRNA‐mediated knockdown of PLP expression that promotes the formation of MBP+ myelin‐like sheets. This finding suggests that RNAi technology is in principle applicable to improve CNS myelination when compromised by PLP/DM20 overexpression. © 2010 Wiley‐Liss, Inc.     

Rab27b is Involved in Lysosomal Exocytosis and Proteolipid Protein Trafficking in Oligodendrocytes

Myelination by oligodendrocytes in the central nervous system requires coordinated exocytosis and endocytosis of the major myelin protein, proteolipid protein(PLP). Here, we demonstrated that a small GTPase,Rab27b, is involved in PLP trafficking in oligodendrocytes.We showed that PLP co-localized with Rab27b in late endosomes/lysosomes in oligodendrocytes. Short hairpinmediated knockdown of Rab27b not only reduced lysosomal exocytosis but also greatly diminished the surface expression of PLP in oligodendrocytes. In addition,knockdown of Rab27b reduced the myelin-like membranes induced by co-culture of oligodendrocytes and neurons.Our data suggest that Rab27b is involved in myelin biogenesis by regulating PLP transport from late endosomes/lysosomes to the cell membrane in oligodendrocytes.     

Microtubule associated protein (MAP1B) is present in cultured oligodendrocytes and co-localizes with tubulin

Differentiation of oligodendrocytes is accompanied by the extension of processes and the assembly of the myelin membrane. It is likely that the cytoskeleton plays an important role in this process in terms of changes in cell shape, transport of myelin components, and organization of the myelin membrane. Oligodendrocytes contain microtubules (MT) which associate with other components of the cytoskeleton, and microtubule associated proteins (MAPs) may mediate some of these interactions. In this study we have shown the presence of MAP1B in oligodendrocytes grown in primary glial cultures by double-label immunofluorescence using antibodies to galactocerebroside (GC) and MAP1B. The staining of the cultures showed that GC-positive oligodendrocytes were also stained with MAP1B antibodies. However, MAP1B stain was limited to cell bodies and processes, whereas GC stain was also seen in flattened membrane sheets and punctate staining in processes. MAP1B staining was also compared with that of myelin proteolipid (PLP), myelin basic protein (MBP) and beta-tubulin in secondary glial cultures that were enriched for oligodendrocytes. The results showed a typical staining of cell bodies and membranous profiles using PLP antibodies, and the staining of cell bodies and flattened regions of membranous sheets by MBP antibodies. In contrast, both polyclonal and monoclonal antibodies to MAP1B showed a uniform diffuse staining of cell bodies, major processes, and fine interconnected processes. Double-labeling of the cells showed that MAP1B was co-localized with tubulin, but was not present in glial fibrillary acidic protein (GFAP)-positive astrocytes. Western and Northern blot analyses of primary glial cultures showed that MAP1B had a molecular mass of 320 kDa and a mRNA of 10 kb. These values are identical to those previously reported for brain MAP1B (Safaei and Fischer, 1989) and demonstrate the presence of MAP1B in oligodendrocytes.     

Lovastatin induces the formation of abnormal myelin-like membrane sheets in primary oligodendrocytes

Statins, well-known inhibitors of cholesterol synthesis and protein isoprenylation, have been proposed as therapeutic drugs for multiple sclerosis (MS). As lovastatin and simvastatin, which are currently tested for their use in MS, can cross the blood-brain barrier, they may affect cellular processes in the central nervous system. This is especially relevant with respect to remyelination as a proposed additional treatment for MS, because cholesterol is a major component of myelin. Here, we show that primary oligodendrocytes, treated with lovastatin, form extensive membrane sheets, which contain galactosphingolipids. However, these membrane sheets are devoid of the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP). Reduced MBP protein expression was confirmed by SDS-PAGE and Western blotting, and in situ hybridization experiments revealed that lovastatin blocks MBP mRNA transport into oligodendrocyte processes. In contrast, PLP expression was only mildly affected by lovastatin. However, lovastatin treatment resulted in intracellular accumulation of PLP and prevented its translocation to the cell surface. Interestingly, another inhibitor of cholesterol synthesis (ro48-8071), which does not interfere with isoprenylation, had a similar effect on the localization of PLP, but it did not affect MBP expression and localization. These results suggest that lovastatin affects PLP transport predominantly by the inhibition of cholesterol synthesis, whereas reduced MBP expression is caused by impaired isoprenylation. Based on these results we recommend to carefully monitor the effect of statins on myelination prior to their use in demyelinating diseases.     

Basic fibroblast growth factor down-regulates myelin basic protein gene expression and alters myelin compaction of mature oligodendrocytes in vitro

The effects of basic fibroblast growth factor (bFGF) on myelin basic protein (MBP) gene expression and myelin-like membrane formation were investigated in oligodendrocyte cultures containing mainly mature oligodendrocytes expressing MBP. These cultures were obtained by selective detachment of the cells of the oligodendrocyte lineage from 40-day-old mixed cultures derived from newborn rat brain. They were further purified by a 3-day pretreatment with cytosine arabinoside (ARA-C) in order to kill cycling cells. After withdrawal of ARA-C, daily treatment of the cells with bFGF for 3 days induced a drastic decrease in MBP mRNA level compared to control cultures treated only with ARA-C. Moreover, the percentage of oligodendrocytes labelled with anti-MBP antibodies decreased by 50%, as well as that of oligodendrocytes expressing myelin oligodendrocyte glycoprotein (MOG), whereas proteolipid protein (PLP) immunolabelled cells were less affected. At the ultrastructural level, myelin-like membranes were still abundant in the ARA-C-and bFGF-treated cultures, but they were conspicuously uncompacted compared to cultures only pretreated with ARA-C. These results bring the first evidence that bFGF is able to down-regulate myelin protein gene expression in mature oligodendrocytes and to alter myelin structure. They imply that if bFGF is secreted after a demyelinating lesion of the central nervous system (CNS), this plasticity of mature oligodendrocytes will allow final remyelination of axons to complete only after this factor has returned to low levels. © 1995 Wiley-Liss, Inc.     

Myelin proteolipid proteins promote the interaction of oligodendrocytes and axons

Although proteolipid protein (PLP) and its DM20 isoform are the major membrane proteins of CNS myelin, their absence causes surprisingly few developmental defects. In comparison, missense mutations of the X-linked Plp gene cause severe dysmyelination. Previous studies have established roles for PLP/DM20 in the formation of the intraperiod line and in maintaining axonal integrity. We now show that a normal number of oligodendrocytes are present in mice lacking PLP/DM20. However, in heterozygous females, which are natural chimeras for X-linked genes, oligodendrocytes lacking PLP/DM20 are in direct competition with wild-type oligodendrocytes that have a distinct advantage. PLP+ oligodendrocytes and PLP+ myelin sheaths make up the greater majority, and this feature is generalised in the CNS throughout life. Moreover, in the absence of PLP/DM20, a proportion of small-diameter axons fails to myelinate, remaining ensheathed but lacking a compact sheath, or show delayed myelination. These findings suggest that PLP/DM20 is also involved in the early stages of axon-oligodendrocyte interaction and wrapping of the axon.     

Proteolipid protein cannot replace P0 protein as the major structural protein of peripheral nervous system myelin

The central nervous system (CNS) of terrestrial vertebrates underwent a prominent molecular change when proteolipid protein (PLP) replaced P₀ protein as the most abundant protein of CNS myelin. However, PLP did not replace P₀ in peripheral nervous system (PNS) myelin. To investigate the possible consequences of a PLP to P₀ shift in PNS myelin, we engineered mice to express PLP instead of P₀ in PNS myelin (PLP‐PNS mice). PLP‐PNS mice had severe neurological disabilities and died between 3 and 6 months of age. Schwann cells in sciatic nerves from PLP‐PNS mice sorted axons into one‐to‐one relationships but failed to form myelin internodes. Mice with equal amounts of P₀ and PLP had normal PNS myelination and lifespans similar to wild‐type (WT) mice. When PLP was overexpressed with one copy of the P₀ gene, sciatic nerves were hypomyelinated; mice displayed motor deficits, but had normal lifespans. These data support the hypothesis that while PLP can co‐exist with P₀ in PNS myelin, PLP cannot replace P₀ as the major structural protein of PNS myelin. GLIA 2015;63:66–77     

Myelination in the absence of galactolipids and proteolipid proteins.

The galactolipids galactocerebroside and sulfatide and the proteolipid protein (PLP) and its splice variant DM20 are the most abundant lipid and protein components of central nervous system myelin. Recent studies have found that mice lacking either the galactolipids or PLP are able to form myelin sheaths with apparently normal periodicity and near normal compaction. Here, we have generated galactolipid/proteolipid double mutants to examine the possibility that these molecules have overlapping functions. We show that the absence of the galactolipids and PLP has pleotropic effects on myelin formation. While oligodendrocytes in the postnatal day 20 galactolipid/proteolipid-deficient mouse are able to elaborate myelin with close to normal intraperiod lines, there is an increased frequency of uncompacted myelin sheaths as well as unmyelinated axons. Moreover, the double mutants display extensive white matter vacuolization of the cerebellum that initiates around postnatal day 16, which correlates with the onset of a severe ataxic phenotype and an increased percentage of apoptotic nuclei in the cerebellar internal granule cell layer. These data indicate that the galactolipids and PLP/DM20 are not required for intraperiod line formation, but they suggest a role for these molecules in mediating myelin compaction and in maintaining the integrity of the cerebellum.     

Identification of antibodies in anti-CNS and anti-PNS myelin sera by immunoblot, characterization by immunohistochemistry, and their effect in tissue culture

Immunoblot analysis of antiserum to rat central nervous system (CNS) myelin revealed antibodies to myelin basic protein (MBP), proteolipid protein (PLP), and numerous high molecular weight proteins. In addition, anti-CNS myelin serum exclusively immunostained 4 basic proteins of rat peripheral nervous system (PNS) myelin. Similarly, anti-PNS myelin sera immunostained many high molecular weight proteins in both CNS and PNS myelin in addition to P0 and 4 basic proteins. Purified MBP and PLP were immunostained by anti-CNS myelin sera and MBP and P0 by anti-PNS myelin sera, indicating that antigenic sites are preserved during protein purification. Immunohistochemical localization with antisera was confined to the myelin sheath except that antisera to CNS myelin also stained oligodendrocytes during the active period of myelination. While anti-CNS myelin sera specifically demyelinated centrally myelinated fibers in culture, none of the anti-PNS myelin sera used here demyelinated organotypic spinal cord-dorsal root ganglion cultures.     

Caspase-3 activation in oligodendrocytes from the myelin-deficient rat

The myelin-deficient (MD) rat has a point mutation in its proteolipid protein (PLP) gene that causes severe dysmyelination and oligodendrocyte cell death. Using an in vitro model, we have shown that MD oligodendrocytes initially differentiate similarly to wild-type cells, expressing galactocerebroside, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin basic protein. However, at the time when PLP expression would normally begin, the MD oligodendrocytes die via an apoptotic pathway involving caspase activation. The active form of caspase-3 was detected, along with the cleavage products of poly-(ADP-ribose) polymerase (PARP) and spectrin, major targets of caspase-mediated proteolysis. A specific inhibitor of casapse-3, Ac-DEVD-CMK, reduced apoptosis in MD oligodendrocytes, but the rescued cells did not mature fully or express myelin-oligodendrocyte glycoprotein. These results suggest that mutant PLP affects not only cell death but also oligodendrocyte differentiation.     

Modification of Schwann cell phenotype with Plp transgenes: Evidence that the PLP and DM20 isoproteins are targeted to different cellular domains

The X-linked proteolipid protein (Plp) gene encodes PLP, the major protein of central nervous system myelin, and its alternative RNA splice product, termed DM20. Schwann cells also express the Plp gene but, in contrast to oligodendrocytes, neither protein is incorporated into peripheral myelin. In the present study, we use different transgenes encoding PLP and DM20 to modify the expression of these proteins in myelin-forming Schwann cells of wild-type and jimpy mice. Increasing the level of PLP, either singly or in combination with DM20, leads to the incorporation of PLP into the compacted myelin sheath; however, DM20 always remains restricted to cytoplasmic regions of the Schwann cell. The insertion of PLP into the membrane does not appear to depend on a cooperativity of the two isoproteins. The presence of PLP does not visibly alter the ultrastructure and periodicity of peripheral nervous system (PNS) myelin. The results indicate that the absence of PLP in the peripheral myelin of normal animals most probably reflects the very low amounts of this isoprotein synthesised by Schwann cells. The preferential incorporation of PLP, as opposed to DM20, in peripheral myelin may indicate that a myelin targeting signal is present in the PLP-specific region of the molecule. J. Neurosci. Res. 50:13–22, 1997. © 1997 Wiley-Liss, Inc.     

Immunohistochemical study of myelin sheaths formed by oligodendrocytes interacting with dissociated dorsal root ganglion neurons in culture

The addition of central nervous system (CNS) glial cells to dissociated networks of rat dorsal root ganglion neurons in tissue culture provided a useful system for the study of CNS myelin sheath formation. The CNS myelin basic proteins (BP) and proteolipid protein (PLP) were demonstrable in these cultures by immunoperoxidase techniques. Both BP and PLP were detectable in myelinating oligodendrocytes and CNS myelin sheaths. Anti-BP serum and anti-PLP serum were useful immunohistochemical staining reagents for the identification of myelinating oligodendrocytes and CNS myelin sheaths in tissue culture.     

Evolution of myelin proteolipid proteins: gene duplication in teleosts and expression pattern divergence

The coevolution of neurons and their supporting glia to the highly specialized axon-myelin unit included the recruitment of proteolipids as neuronal glycoproteins (DMbeta, DMgamma) or myelin proteins (DMalpha/PLP/DM20). Consistent with a genome duplication at the root of teleosts, we identified three proteolipid pairs in zebrafish, termed DMalpha1 and DMalpha2, DMbeta1 and DMbeta2, DMgamma1 and DMgamma2. The paralogous amino acid sequences diverged remarkably after gene duplication, indicating functional specialization. Each proteolipid has adopted a distinct spatio-temporal expression pattern in neural progenitors, neurons, and in glia. DMalpha2, the closest homolog to mammalian PLP/DM20, is coexpressed with P0 in oligodendrocytes and upregulated after optic nerve lesion. DMgamma2 is expressed in multipotential stem cells, and the other four proteolipids are confined to subsets of CNS neurons. Comparing protein sequences and gene structures from birds, teleosts, one urochordate species, and four invertebrates, we have reconstructed major steps in the evolution of proteolipids.     

Site-specific antibodies to rat myelin proteolipids directed against the C-terminal hexapeptide

A site-specific antiserum against the rat myelin proteolipids was produced in rabbits by injection of a synthetic polypeptide composed of the C-terminal amino acids of the proteolipid sequence. The immunogenic hexapeptide H-Gly-Arg-Gly-Thr-Lys-Phe-OH was coupled to chicken egg-albumin with dimethylsuberimidate. Antibodies specific for this peptide reacted with the 2 myelin proteolipid protein bands after SDS polyacrylamide gel electrophoresis and electrophoretic transfer onto nitrocellulose. Immunocytochemical investigations with this anti-peptide antiserum showed that the Golgi complexes of the oligodendrocytes were highly labeled as noted previously with multivalent antibodies. Labeling of vesicles and discontinuous staining of the plasmalemma were also observed in the most actively myelinating oligodendrocytes. In contrast to previous results, the major dense line was free of staining; this may indicate that at this site the C-terminal hexapeptide is inaccessible to these antibodies and perhaps buried in the lipid bilayer, in disagreement with the proposed organization of the myelin proteolipid in the myelin membrane.     

Differential sensitivity in the survival of oligodendrocyte cell lines to overexpression of myelin proteolipid protein gene products

The proteolipid (PLP) gene encodes at least four proteins, including the classic PLP and DM20, which are important components of the myelin sheath, and the recently identified soma-restricted (sr) isoforms, srPLP and srDM20. The classic PLP and DM20 gene products have been implicated in oligodendrocyte survival by overexpression studies in vitro and in vivo. The classic and sr proteolipids are targeted to different cellular compartments in the oligodendrocyte, suggesting different cellular functions. Accordingly, we examined the effects of in vitro overexpression of the sr-PLP/DM20 isoforms on the survival of stably transfected, conditionally immortalized, oligodendroglial cell lines and compared this to overexpression of the classic and the jimpy-mutated proteolipids. The results indicate that overexpression of either normal or jimpy classic PLP/DM20 resulted in a dramatic reduction in the survival of the oligodendrocyte cell lines at the nonpermissive temperature, but not the COS-7 cell line, a cell line expressing the same oncogene constitutively. Survival of the oligodendrocyte cell lines was significantly less affected when either the sr-PLP/DM20 or the dopamine D-2 receptor, another cell membrane protein, was overexpressed in the cell lines. These results suggest that overexpression of the "classic" PLP or DM20 can compromise the survival of oligodendrocytes whether or not they are mutated. Furthermore, they suggest that the internal mechanisms for normal targeting of the PLP/DM20 isoforms of either the "classic" or the "sr" types influence the oligodendrocyte's ability to survive when these proteolipids are overexpressed.