Paul Montague

Oligodendroglial modulation of fast axonal transport in a mouse model of hereditary spastic paraplegia.

Oligodendrocytes are critical for the development of the plasma membrane and cytoskeleton of the axon. In this paper, we show that fast axonal transport is also dependent on the oligodendrocyte. Using a mouse model of hereditary spastic paraplegia type 2 due to a null mutation of the myelin Plp gene, we find a progressive impairment in fast retrograde and anterograde transport. Increased levels of retrograde motor protein subunits are associated with accumulation of membranous organelles distal to nodal complexes. Using cell transplantation, we show categorically that the axonal phenotype is related to the presence of the overlying Plp null myelin. Our data demonstrate a novel role for oligodendrocytes in the local regulation of axonal function and have implications for the axonal loss associated with secondary progressive multiple sclerosis.

FGF/heparin differentially regulates Schwann cell and olfactory ensheathing cell interactions with astrocytes: a role in astrocytosis

After injury, the CNS undergoes an astrocyte stress response characterized by reactive astrocytosis/proliferation, boundary formation, and increased glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycan (CSPG) expression. Previously, we showed that in vitro astrocytes exhibit this stress response when in contact with Schwann cells but not olfactory ensheathing cells (OECs). In this study, we confirm this finding in vivo by demonstrating that astrocytes mingle with OECs but not Schwann cells after injection into normal spinal cord. We show that Schwann cell-conditioned media (SCM) induces proliferation in monocultures of astrocytes and increases CSPG expression in a fibroblast growth factor receptor 1 (FGFR1)-independent manner. However, SCM added to OEC/astrocyte cocultures induces reactive astrocytosis and boundary formation, which, although sensitive to FGFR1 inhibition, was not induced by FGF2 alone. Addition of heparin to OEC/astrocyte cultures induces boundary formation, whereas heparinase or chlorate treatment of Schwann cell/astrocyte cultures reduces it, suggesting that heparan sulfate proteoglycans (HSPGs) are modulating this activity. In vivo, FGF2 and FGFR1 immunoreactivity was increased over grafted OECs and Schwann cells compared with the surrounding tissue, and HSPG immunoreactivity is increased over reactive astrocytes bordering the Schwann cell graft. These data suggest that components of the astrocyte stress response, including boundary formation, astrocyte hypertrophy, and GFAP expression, are mediated by an FGF family member, whereas proliferation and CSPG expression are not. Furthermore, after cell transplantation, HSPGs may be important for mediating the stress response in astrocytes via FGF2. Identification of factors secreted by Schwann cells that induce this negative response in astrocytes would further our ability to manipulate the inhibitory environment induced after injury to promote regeneration.

The proteolipid protein gene

Proteolipid protein (PLP) is the major myelin protein of the CNS and is believed to have a structural role in maintaining the intraperiod line of compact myelin. An isoform. DM‐20. produced by alternative splicing of exon 3B is expressed earlier than PLP in the CNS and may be involved in glial cell development. DM‐20 is also present in myelin‐forming and non‐myelin‐forming Schwann cells, olfactory nerve ensheathing cells, some glial cell lines and cardiac myocytes. Molecular studies suggest the existence of a PLP gene family with sequence similarities between molecules of different species. Such studies also lend credence to the suggestion that PLP and/or DM‐20 may function as a membrane pore. Mutations in the PLP gene occur in several animal species and cause severe pleiotropic effects on myelination. In man this presents as Pelizaeus‐Merzbacher disease (PMD). The phenotype of such mutants is characterized by dysmyelination with myelin of abnormal periodicity, paucity of mature oligodendrocytes and astrocytosis. Duplication of the PLP gene in transgenic animals or in one form of PMD also results in dysmyelination. X‐linked spastic paraplegia (SPG2) is allelic to PMD and is associated with PLP mutations in which the levels of the DM‐20 isoform are probably relatively normal. The effects of PLP gene dosage on CNS myelination can be compared in many ways to the variety of phenotypes in the PNS in hereditary neuropathies of the Charcot‐Marie‐Tooth type in which the peripheral myelin‐22 gene is mutated.

Phenotypic severity of murine Plp mutants reflects in vivo and in vitro variatioans in transport of PLP isoproteins

Mutations of the major myelin gene, proteolipid protein (Plp), cause Pelizaeus‐Merzbacher disease and some forms of spastic paraplegia in man and dysmyelinating phenotypes in animals. The clinical severity is markedly heterogeneous, ranging from relatively mild to severe and fatal. Point mutations, or frame shifts, which are predicted to result in translation of structurally altered proteins account for many of these cases, including 3 of the allelic murine conditions. Plpjp‐rsh, Plpjp‐msd, and Plpjp represent an increasing severity of clinical and pathological phenotypes, respectively. In this study we determined whether there was any correlation between the severity of phenotype and the transport of the predicted abnormal protein. We examined the ability of the two products of the Plp gene, PLP and DM20, to insert into the plasma membrane of transfected BHK or COS‐7 cells, and into the myelin sheath of oligodendrocytes. With these complementary in vitro and in vivo approaches we find that proteins of Plpjp‐rsh, associated with the mildest phenotype, have a far greater ability to insert into the cell membrane or myelin than those associated with the severe phenotypes. Additionally, altered DM20 is more readily transported to the cell surface and to myelin than the PLP isoprotein. Interestingly, the two clonal cell lines chosen for transient transfection differ in their ability to fold DM20 from Plpjp‐rsh and Plpjp‐msd mice correctly, as inferred by staining for the conformation‐sensitive O10 epitope. In the case of Plpjp, which is associated with the most severe phenotype, no PLP or O10 staining is present at the cell surface or in myelin. The perturbation in trafficking observed for altered Plpjp PLP and DM20 in oligodendrocytes does not extend to other myelin membrane proteins, such as MAG and MOG, nor to wild type PLP co‐expressed in the same cell, all of which are correctly inserted into myelin. As Plp‐knockout mice do not have a dysmyelinating phenotype it seems unlikely that absence of PLP and/or DM20 in the membrane is responsible for the pathology. It remains to be determined whether the perturbation in protein trafficking is associated with the dysmyelination, or if the altered product of the mutant alleles acquire a novel function which is deleterious to myelin production by oligodendrocytes. GLIA 20:322–332, 1997.

Oligodendrocyte progenitors in the embryonic spinal cord express DM-20.

Oligodendrocyte progenitors, originating in the ventral ventricular zone of the embryonic rodent spinal cord, migrate and differentiate into the oligodendrocytes myelinating the future white matter. Transcripts for the dm-20 isoform of the proteolipid protein (plp) gene are detectable initially in cells of the ventral ventricular region of the embryonic central canal and subsequently throughout the white matter. The dm-20+ cells are present several days before oligodendrocytes or myelin sheaths are detectable. The purpose of the present study was to determine if DM-20 protein is present and whether DM-20+ cells can be linked to the oligodendrocyte lineage in the mouse spinal cord. Expression of plp and dm-20 transcripts and product was monitored using reverse transcription polymerase chain reaction (RT-PCR), and in situ hybridization and immunostaining of cryosections and associated cultures. Cell identification was performed using antigenic markers characterizing different stages of oligodendrocyte differentiation. We show a temporal and spatial progression of cells expressing dm-20 transcripts and product from the ventral ventricular zone at embryonic day 13 (E13.0), via the lateral borders of the floor plate to the ventral pia and white matter. The cells, initially devoid of myelin basic protein (MBP) and PLP, co-express these myelin proteins at approximately E16.5/17.0. Some DM-20+ cells co-label with definitive markers of the early oligodendrocyte lineage, are capable of mitosis and subsequently differentiate into oligodendrocytes. Other DM-20+ cells may represent earlier precursor cells. The expression of DM-20 in oligodendrocyte progenitors is consistent with a postulated role in glial cell development.

Genetic background determines phenotypic severity of the Plp rumpshaker mutation

The rumpshaker mutation of the proteolipid protein (Plp) gene causes dysmyelination in man and mouse. We show that the phenotype in the mouse depends critically on the genetic background in which the mutation is expressed. On the C3H background there is normal longevity whereas changing to a C57BL/6 strain results in seizures and death at around postnatal day 30. The more severe phenotype is associated with less myelin and reduced levels of major myelin proteins. There are also more apoptotic cells, including oligodendrocytes, increased numbers of proliferating cells, increased numbers of NG2+ oligodendrocyte progenitors and increased microglia compared to the milder phenotype. The number of mature oligodendrocytes is similar to wild‐type in both strains of mutant, however, suggesting that increased oligodendrocyte death is matched by increased generation from progenitors. The dichotomy of phenotype probably reflects the influence of modifying loci. The localization of these putative modifying genes and their mode of action remain to be determined.

PLP overexpression perturbs myelin protein composition and myelination in a mouse model of Pelizaeus-Merzbacher disease

Duplication of PLP1, an X‐linked gene encoding the major myelin membrane protein of the human CNS, is the most frequent cause of Pelizaeus‐Merzbacher disease (PMD). Transgenic mice with extra copies of the wild type Plp1 gene, a valid model of PMD, also develop a dysmyelinating phenotype dependant on gene dosage. In this study we have examined the effect of increasing Plp1 gene dosage on levels of PLP/DM20 and on other representative myelin proteins. In cultured oligodendrocytes and early myelinating oligodendrocytes in vivo, increased gene dosage leads to elevated levels of PLP/DM20 in the cell body. During myelination, small increases in Plp1 gene dosage (mice hemizygous for the transgene) elevate the level of PLP/DM20 in oligodendrocyte soma but cause only minimal and transient effects on the protein composition and structure of myelin suggesting that cells can regulate the incorporation of proteins into myelin. However, larger increases in dosage (mice homozygous for the transgene) are not well tolerated, leading to hypomyelination and alteration in the cellular distribution of PLP/DM20. A disproportionate amount of PLP/DM20 is retained in the cell soma, probably in autophagic vacuoles and lysosomes whereas the level in myelin is reduced. Increased Plp1 gene dosage affects other myelin proteins, particularly MBP, which is transitorily reduced in hemizygous mice but consistently and markedly lower in homozygotes in both myelin and naïve or early myelinating oligodendrocytes. Whether the reduced MBP is implicated in the pathogenesis of dysmyelination is yet to be established.

Expression of the proteolipid protein gene in glial cells of the post‐natal peripheral nervous system of rodents

The proteolipid protein (PLP) gene encodes for two proteins, PLP and DM‐20, which are produced by alternative splicing of exon 3B. PLP is the major CNS myelin protein and is postulated to play a structural role at the intraperiod line. Its developmental expression mirrors that of CNS myelination. DM‐20 predominates in the embryo and prior to myelination of the CNS and may be involved in glial cell development. The PLP gene is expressed in the PNS in which DM‐20 is the predominant isoform at all ages. In this study we describe the localization of the two isoforms in the post‐natal rodent PNS using immunostaining and reverse transcriptase PCR. DM‐20 is present in relatively high abundance in non‐myelin‐forming Schwann cells and within cytoplasmic regions of myelinated internodes, particularly the paranodes and Schmidt‐Lanterman incisures and also the outer Schwann cytoplasm and perinuclear cytoplasm. DM‐20 is also located in the perineuronal satellite cells of spinal, cranial and autonomic ganglia and in the ensheathing cells of the olfactory nerve layer of the olfactory bulb. PLP was detected by immunocyto‐chemistry in the perinuclear region of myelinated inter‐nodes: PCR analysis indicated small amounts of PLP mRNA in the other locations but protein was not detected by immunostaining. Neither protein was identified in compact myelin of the PNS. DM‐20 is the predominant product of the PLP gene expressed in a wide variety of peripheral glia. Its presence is not correlated to a myelin‐forming state. Other studies have demonstrated early embryonic expression of the PLP gene throughout the PNS and all these features support the hypothesis that any putative role for DM‐20 is unrelated to myelination but may involve glial cell development.

Demyelination and axonal preservation in a transgenic mouse model of Pelizaeus-Merzbacher disease

It is widely thought that demyelination contributes to the degeneration of axons and, in combination with acute inflammatory injury, is responsible for progressive axonal loss and persistent clinical disability in inflammatory demyelinating disease. In this study we sought to characterize the relationship between demyelination, inflammation and axonal transport changes using a Plp1‐transgenic mouse model of Pelizaeus‐Merzbacher disease. In the optic pathway of this non‐immune mediated model of demyelination, myelin loss progresses from the optic nerve head towards the brain, over a period of months. Axonal transport is functionally perturbed at sites associated with local inflammation and ‘damaged’ myelin. Surprisingly, where demyelination is complete, naked axons appear well preserved despite a significant reduction of axonal transport. Our results suggest that neuroinflammation and/or oligodendrocyte dysfunction are more deleterious for axonal health than demyelination per se, at least in the short term.

Myelin-Associated Oligodendrocytic Basic Protein: A Family of Abundant CNS Myelin Proteins in Search of a Function

The myelin-associated oligodendrocytic basic protein (MOBP) family constitutes the third most abundant protein in CNS myelin. The mouse Mobp gene comprises eight exons. Mobp pre-mRNA processing gives rise to at least seven Mobp splice variants which are expressed solely in the oligodendrocyte. The predicted proteins all, with one exception, share a 68 residue amino terminus, encoded by exon 3. The carboxyl termini differ in length, giving rise to the diverse array of the protein isoforms. Like myelin basic protein, MOBP is present in the major dense line of CNS myelin suggesting a role in the compaction or stabilization of myelin. However, Mobp homozygous null mice display no overt clinical phenotype and no defect in the process of myelination. MOBP can induce experimental allergic encephalomyelitis in mice and has been proposed to have a role in the pathogenesis of multiple sclerosis. Despite 10 years of rigorous study, the normal physiological function of MOBP remains unknown.