PLP1 and CNTN1 gene variation modulates the microstructure of human white matter in the corpus callosum.

The corpus callosum is the brain's largest commissural fiber tract and is crucial for interhemispheric integration of neural information. Despite the high relevance of the corpus callosum for several cognitive systems, the molecular determinants of callosal microstructure are largely unknown. Recently, it was shown that genetic variations in the myelin-related proteolipid 1 gene PLP1 and the axon guidance related contactin 1 gene CNTN1 were associated with differences in interhemispheric integration at the behavioral level. Here, we used an innovative new diffusion neuroimaging technique called neurite orientation dispersion and density imaging (NODDI) to quantify axonal morphology in subsections of the corpus callosum and link them to genetic variation in PLP1 and CNTN1. In a cohort of 263 healthy human adults, we found that polymorphisms in both PLP1 and CNTN1 were significantly associated with callosal microstructure. Importantly, we found a double dissociation between gene function and neuroimaging variables. Our results suggest that genetic variation in the myelin-related gene PLP1 impacts white matter microstructure in the corpus callosum, possibly by affecting myelin structure. In contrast, genetic variation in the axon guidance related gene CNTN1 impacts axon density in the corpus callosum. These findings suggest that PLP1 and CNTN1 gene variations modulate specific aspects of callosal microstructure that are in line with their gene function.

Striking parallels between carotid body glomus cell and adrenal chromaffin cell development.

Carotid body glomus cells mediate essential reflex responses to arterial blood hypoxia. They are dopaminergic and secrete growth factors that support dopaminergic neurons, making the carotid body a potential source of patient-specific cells for Parkinson's disease therapy. Like adrenal chromaffin cells, which are also hypoxia-sensitive, glomus cells are neural crest-derived and require the transcription factors Ascl1 and Phox2b; otherwise, their development is little understood at the molecular level. Here, analysis in chicken and mouse reveals further striking molecular parallels, though also some differences, between glomus and adrenal chromaffin cell development. Moreover, histology has long suggested that glomus cell precursors are 'émigrés' from neighbouring ganglia/nerves, while multipotent nerve-associated glial cells are now known to make a significant contribution to the adrenal chromaffin cell population in the mouse. We present conditional genetic lineage-tracing data from mice supporting the hypothesis that progenitors expressing the glial marker proteolipid protein 1, presumably located in adjacent ganglia/nerves, also contribute to glomus cells. Finally, we resolve a paradox for the 'émigré' hypothesis in the chicken - where the nearest ganglion to the carotid body is the nodose, in which the satellite glia are neural crest-derived, but the neurons are almost entirely placode-derived - by fate-mapping putative nodose neuronal 'émigrés' to the neural crest.

Experimental Autoimmune Encephalomyelitis (EAE) as Animal Models of Multiple Sclerosis (MS).

Multiple sclerosis (MS) is a multifocal demyelinating disease of the central nervous system (CNS) leading to the progressive destruction of the myelin sheath surrounding axons. It can present with variable clinical and pathological manifestations, which might reflect the involvement of distinct pathogenic processes. Although the mechanisms leading to the development of the disease are not fully understood, numerous evidences indicate that MS is an autoimmune disease, the initiation and progression of which are dependent on an autoimmune response against myelin antigens. In addition, genetic susceptibility and environmental triggers likely contribute to the initiation of the disease. At this time, there is no cure for MS, but several disease-modifying therapies (DMTs) are available to control and slow down disease progression. A good number of these DMTs were identified and tested using animal models of MS referred to as experimental autoimmune encephalomyelitis (EAE). In this review, we will recapitulate the characteristics of EAE models and discuss how they help shed light on MS pathogenesis and help test new treatments for MS patients.

MmpL transporter-mediated export of cell-wall associated lipids and siderophores in mycobacteria.

Mycobacteria produce a large variety of surface-exposed lipids with unusual structures. Some of these compounds are ubiquitously present in mycobacteria and play an important role in the structural organization of the cell envelope, while others are species-specific. The biosynthesis of most of these lipids requires modular polyketide synthases (PKS) or non-ribosomal peptide synthetases (NRPS) that are intracellular, suggesting that the assembly of these compounds takes place in the cytosolic compartment or near the inner leaflet of the plasma membrane. The molecular mechanisms that mediate the export of these lipid components across the cell envelope remain poorly understood. Mycobacterial membrane protein Large (MmpL) transporters, a subclass of Resistance-Nodulation-Cell Division (RND) transporters, appear to play a major role in this process, acting as scaffold proteins that couple lipid synthesis and transport. Recent studies have shown that this family of transporters also contributes to siderophore secretion in Mycobacterium tuberculosis. The goal of this review is to provide the most recent advances in our understanding of the molecular mechanisms involved in lipid and siderophore transport mediated by MmpL transporters.

The major myelin-resident protein PLP is transported to myelin membranes via a transcytotic mechanism: involvement of sulfatide.

Myelin membranes are sheet-like extensions of oligodendrocytes that can be considered membrane domains distinct from the cell's plasma membrane. Consistent with the polarized nature of oligodendrocytes, we demonstrate that transcytotic transport of the major myelin-resident protein proteolipid protein (PLP) is a key element in the mechanism of myelin assembly. Upon biosynthesis, PLP traffics to myelin membranes via syntaxin 3-mediated docking at the apical-surface-like cell body plasma membrane, which is followed by subsequent internalization and transport to the basolateral-surface-like myelin sheet. Pulse-chase experiments, in conjunction with surface biotinylation and organelle fractionation, reveal that following biosynthesis, PLP is transported to the cell body surface in Triton X-100 (TX-100)-resistant microdomains. At the plasma membrane, PLP transiently resides within these microdomains and its lateral dissipation is followed by segregation into 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)-resistant domains, internalization, and subsequent transport toward the myelin membrane. Sulfatide triggers PLP's reallocation from TX-100- into CHAPS-resistant membrane domains, while inhibition of sulfatide biosynthesis inhibits transcytotic PLP transport. Taking these findings together, we propose a model in which PLP transport to the myelin membrane proceeds via a transcytotic mechanism mediated by sulfatide and characterized by a conformational alteration and dynamic, i.e., transient, partitioning of PLP into distinct membrane microdomains involved in biosynthetic and transcytotic transport.

Myelin architecture: zippering membranes tightly together.

Rapid nerve conduction requires the coating of axons by a tightly packed multilayered myelin membrane. In the central nervous system, myelin is formed from cellular processes that extend from oligodendrocytes and wrap in a spiral fashion around an axon, resulting in the close apposition of adjacent myelin membrane bilayers. In this review, we discuss the physical principles underlying the zippering of the plasma membrane of oligodendrocytes at the cytoplasmic and extracellular leaflet. We propose that the interaction of the myelin basic protein with the cytoplasmic leaflet of the myelin bilayer triggers its polymerization into a fibrous network that drives membrane zippering and protein extrusion. In contrast, the adhesion of the extracellular surfaces of myelin requires the down-regulation of repulsive components of the glycocalyx, in order to uncover weak and unspecific attractive forces that bring the extracellular surfaces into close contact. Unveiling the mechanisms of myelin membrane assembly at the cytoplasmic and extracelluar sites may help to understand how the myelin bilayers are disrupted and destabilized in the different demyelinating diseases.

A critical role for the cholesterol-associated proteolipids PLP and M6B in myelination of the central nervous system.

The formation of central nervous system myelin by oligodendrocytes requires sterol synthesis and is associated with a significant enrichment of cholesterol in the myelin membrane. However, it is unknown how oligodendrocytes concentrate cholesterol above the level found in nonmyelin membranes. Here, we demonstrate a critical role for proteolipids in cholesterol accumulation. Mice lacking the most abundant myelin protein, proteolipid protein (PLP), are fully myelinated, but PLP-deficient myelin exhibits a reduced cholesterol content. We therefore hypothesized that "high cholesterol" is not essential in the myelin sheath itself but is required for an earlier step of myelin biogenesis that is fully compensated for in the absence of PLP. We also found that a PLP-homolog, glycoprotein M6B, is a myelin component of low abundance. By targeting the Gpm6b-gene and crossbreeding, we found that single-mutant mice lacking either PLP or M6B are fully myelinated, while double mutants remain severely hypomyelinated, with enhanced neurodegeneration and premature death. As both PLP and M6B bind membrane cholesterol and associate with the same cholesterol-rich oligodendroglial membrane microdomains, we suggest a model in which proteolipids facilitate myelination by sequestering cholesterol. While either proteolipid can maintain a threshold level of cholesterol in the secretory pathway that allows myelin biogenesis, lack of both proteolipids results in a severe molecular imbalance of prospective myelin membrane. However, M6B is not efficiently sorted into mature myelin, in which it is 200-fold less abundant than PLP. Thus, only PLP contributes to the high cholesterol content of myelin by association and co-transport.

Increased Plp1 gene expression leads to massive microglial cell activation and inflammation throughout the brain.

PMD (Pelizaeus-Merzbacher disease) is a rare neurodegenerative disorder that impairs motor and cognitive functions and is associated with a shortened lifespan. The cause of PMD is mutations of the PLP1 [proteolipid protein 1 gene (human)] gene. Transgenic mice with increased Plp1 [proteolipid protein 1 gene (non-human)] copy number model most aspects of PMD patients with duplications. Hypomyelination and demyelination are believed to cause the neurological abnormalities in mammals with PLP1 duplications. We show, for the first time, intense microglial reactivity throughout the grey and white matter of a transgenic mouse line with increased copy number of the native Plp1 gene. Activated microglia in the white and grey matter of transgenic mice are found as early as postnatal day 7, before myelin commences in normal cerebra. This finding indicates that degeneration of myelin does not cause the microglial response. Microglial numbers are doubled due to in situ proliferation. Compared with the jp (jimpy) mouse, which has much more oligodendrocyte death and hardly any myelin, microglia in the overexpressors show a more dramatic microglial reactivity than jp, especially in the grey matter. Predictably, many classical markers of an inflammatory response, including TNF-α (tumour necrosis factor-α) and IL-6, are significantly up-regulated manyfold. Because inflammation is believed to contribute to axonal degeneration in multiple sclerosis and other neurodegenerative diseases, inflammation in mammals with increased Plp1 gene dosage may also contribute to axonal degeneration described in patients and rodents with PLP1 increased gene dosage.

Ectopic T-cell specificity and absence of perforin and granzyme B alleviate neural damage in oligodendrocyte mutant mice.

In transgenic mice overexpressing the major myelin protein of the central nervous system, proteolipid protein, CD8+ T-lymphocytes mediate the primarily genetically caused myelin and axon damage. In the present study, we investigated the cellular and molecular mechanisms underlying this immune-related neural injury. At first, we investigated whether T-cell receptors (TCRs) are involved in these processes. For this purpose, we transferred bone marrow from mutants carrying TCRs with an ectopic specificity to ovalbumin into myelin mutant mice that also lacked normal intrinsic T-cells. T-lymphocytes with ovalbumin-specific TCRs entered the mutant central nervous system to a similar extent as T-lymphocytes from wild-type mice. However, as revealed by histology, electron microscopy and axon- and myelin-related immunocytochemistry, these T-cells did not cause neural damage in the myelin mutants, reflecting the need for specific antigen recognition by cytotoxic CD8+ T-cells. By chimerization with bone marrow from perforin- or granzyme B (Gzmb)-deficient mice, we demonstrated that absence of these cytotoxic molecules resulted in reduced neural damage in myelin mutant mice. Our study strongly suggests that pathogenetically relevant immune reactions in proteolipid protein-overexpressing mice are TCR-dependent and mediated by the classical components of CD8+ T-cell cytotoxicity, perforin, and Gzmb. These findings have high relevance with regard to our understanding of the pathogenesis of disorders primarily caused by genetically mediated oligodendropathy.

Proteolipid protein 2 is associated with melanoma metastasis.

The metastasis of malignant tumor cells from the primary tumor to distant sites in the body is a complex process. To identify genes that may be essential for metastasis, we established poorly metastatic mouse melanoma cells, namely Y925F-mutated FAK-transfected cells (Y925F cells), from the highly metastatic mouse melanoma cell line B16F10, and performed expression analyses. The expression of phospholipid protein 2 (PLP2) was markedly down-regulated in the Y925F cells. To elucidate the function of PLP2, we established melanoma cells overexpressing PLP2. We found that PLP2 enhanced proliferation, adhesion, invasion, and MMP-2 secretion in vitro, and tumor metastasis in vivo. These results suggest that PLP2 aids metastasis. Furthermore, we showed that PLP2 binds specifically to PI3K, thus activating Akt.

The myelin proteolipid DMα in fishes.

Vertebrate myelin membranes are compacted and held in close apposition by three structural proteins of myelin, myelin basic protein, myelin protein zero (MPZ) and myelin proteolipid protein (PLP1/DMalpha). PLP1/DMalpha is considered to function as a scaffolding protein and play a role in intracellular trafficking in oligodendrocytes. In humans, point mutations, duplications or deletions of PLP1 are associated with Pelizaeus-Merzbacher disease and spastic paraplegia Type 2. PLP1 is highly conserved between mammals, but less so in lower vertebrates. This has led some researchers to question whether certain fish species express PLP1 orthologues at all, and to suggest that the function of PLP1/DMalpha in the central nervous system (CNS) may have been taken over by MPZ. Here, we review the evidence for the conservation of orthologues of PLP1/DMalpha in actinopterygian fishes and provide a comparison of currently available sequence data across 17 fish species. Our analysis demonstrates that orthologues of PLP1/DMalpha have been retained and are functionally expressed in many, if not all, extant species of bony fish. Many of the amino acids that, when mutated, are associated with severe CNS pathology are conserved in teleosts, demonstrating conservation of essential functions and justifying the development of novel disease models in species such as the zebrafish.

The molecular and cellular defects underlying Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a recessive X-linked dysmyelinating disorder of the central nervous system (CNS). The most frequent cause of PMD is a genomic duplication of chromosome Xq22 including the region encoding the dosage-sensitive proteolipid protein 1 (PLP1) gene. The PLP1 duplications are heterogeneous in size, unlike duplications causing many other genomic disorders, and arise by a distinct molecular mechanism. Other causes of PMD include PLP1 deletions, triplications and point mutations. Mutations in the PLP1 gene can also give rise to spastic paraplegia type 2 (SPG2), an allelic form of the disease. Thus, there is a spectrum of CNS disorder from mild SPG2 to severe connatal PMD. PLP1 encodes a major protein in CNS myelin and is abundantly expressed in oligodendrocytes, the myelinating cells of the CNS. Significant advances in our understanding of PMD have been achieved by investigating mutant PLP1 in PMD patients, animal models and in vitro studies. How the different PLP1 mutations and dosage effects give rise to PMD is being revealed. Interestingly, the underlying causes of pathogenesis are distinct for each of the different genetic abnormalities. This article reviews the genetics of PMD and summarises the current knowledge of causative molecular and cellular mechanisms.

Proteolipid protein is required for transport of sirtuin 2 into CNS myelin.

Mice lacking the expression of proteolipid protein (PLP)/DM20 in oligodendrocytes provide a genuine model for spastic paraplegia (SPG-2). Their axons are well myelinated but exhibit impaired axonal transport and progressive degeneration, which is difficult to attribute to the absence of a single myelin protein. We hypothesized that secondary molecular changes in PLP(null) myelin contribute to the loss of PLP/DM20-dependent neuroprotection and provide more insight into glia-axonal interactions in this disease model. By gel-based proteome analysis, we identified >160 proteins in purified myelin membranes, which allowed us to systematically monitor the CNS myelin proteome of adult PLP(null) mice, before the onset of disease. We identified three proteins of the septin family to be reduced in abundance, but the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin 2 (SIRT2) was virtually absent. SIRT2 is expressed throughout the oligodendrocyte lineage, and immunoelectron microscopy revealed its association with myelin. Loss of SIRT2 in PLP(null) was posttranscriptional, suggesting that PLP/DM20 is required for its transport into the myelin compartment. Because normal SIRT2 activity is controlled by the NAD+/NADH ratio, its function may be coupled to the axo-glial metabolism and the long-term support of axons by oligodendrocytes.

Glutamate stimulates oligodendrocyte progenitor migration mediated via an alphav integrin/myelin proteolipid protein complex.

In the mammalian CNS, oligodendrocyte precursor cells (OPCs) express most neurotransmitter receptors, but their function remains unclear. The current studies suggest a physiological role for glutamate (AMPA and/or kainate) receptors in OPC migration. AMPA stimulated alphav integrin-mediated OPC migration by increasing both the rate of cell movement and the frequency of Ca2+ transients. A protein complex containing the myelin proteolipid protein (PLP) and alphav integrin modulated the AMPA-stimulated migration, and stimulation of OPC AMPA receptors resulted in increased association of the AMPA receptor subunits themselves with the alphav integrin/PLP complex. Thus, after AMPA receptor stimulation, an alphav integrin/PLP/neurotransmitter receptor protein complex forms that reduces binding to the extracellular matrix and enhances OPC migration. To assess the extent to which PLP was involved in the AMPA-stimulated migration, OPCs from the myelin-deficient (MD) rat, which has a PLP gene mutation, were analyzed. OPCs from the MD rat had a normal basal migration rate, but AMPA did not stimulate the migration of these cells, suggesting that the PLP/alphav integrin complex was important for the AMPA-mediated induction. AMPA-induced modulation of OPC migration was abolished by pertussis toxin, although baseline migration was normal. Thus, G-protein-dependent signaling is crucial for AMPA-stimulated migration of OPCs but not for basal OPC migration. Other signaling pathways involved in this AMPA-stimulated OPC migration were also determined. These studies highlight novel signaling determinants of OPC migration and suggest that glutamate could play a pivotal role in regulating integrin-mediated OPC migration.

An immunomodulatory GpG oligonucleotide for the treatment of autoimmunity via the innate and adaptive immune systems.

Bacterial DNA and immunostimulatory CpG oligodeoxynucleotides (ODNs) activate the innate immune system to produce proinflammatory cytokines. Shown to be potent Th1-like adjuvants, stimulatory CpG motifs are currently used as effective therapeutic vaccines for various animal models of infectious diseases, tumors, allergies, and autoimmune diseases. In this study, we show that the application of an immunomodulatory GpG ODN, with a single base switch from CpG to GpG, can effectively inhibit the activation of Th1 T cells associated with autoimmune disease. Moreover, this immunomodulatory GpG ODN suppresses the severity of experimental autoimmune encephalomyelitis in mice, a prototypic Th1-mediated animal disease model for multiple sclerosis.

Characterization of myelination in the developing zebrafish.

Myelination, the process by which glial cells ensheath and electrically insulate axons, has been investigated intensely. Nevertheless, knowledge of how myelination is regulated or how myelinating cells communicate with neurons is still incomplete. As a prelude to genetic analyses of these processes, we have identified zebrafish orthologues of genes encoding major myelin proteins and have characterized myelination in the larval zebrafish. Expression of genes corresponding to proteolipid protein (PLP/DM20), myelin protein zero (P0), and myelin basic protein (MBP) is detected at 2 days postfertilization (dpf), first in the ventral hindbrain, close to the midline. During the next 8 days, expression spreads rostrally to the midbrain and optic nerve, and caudally to the spinal cord. DM20 is expressed in the CNS only, while MBP transcripts are detected both in the CNS and in Schwann cells of the lateral line, cranial nerves, and spinal motor nerves. Unlike its closest homologue, trout IP1, zebrafish P0 transcripts were restricted to the CNS. Ultrastructurally, the expression of myelin genes correlated well with myelination, although myelination showed a temporal lag. Myelinated axons were first detected at 4 dpf in the ventral hindbrain, where they were loosely wrapped by processes of glia cells. By 7 dpf, bundles of heavily myelinated axons were observed in the same region. Axons in the lateral line and optic nerves were also surrounded by compact myelin. Conservation in gene expression patterns and the early appearance of myelinated axons, support using the zebrafish to dissect the process of myelination by a genetic approach.

Myelin proteolipid protein-induced aggregation of lipid vesicles: efficacy of the various molecular species.

The different molecular species that form the myelin proteolipid protein family were isolated by size-exclusion and ion-exchange chromatography in organic solvents and their adhesive properties were tested using a vesicle aggregation assay. Addition of the major proteolipid (PLP) to phosphatidylcholine-cholesterol vesicles caused their clustering as determined by increase in O.D.(450 nm) and by transmission electron microscopy. A small fraction of the aggregated vesicles underwent fusion as determined by resonance energy transfer experiments. Vesicle aggregation by PLP, but not the dissociation of the aggregates, was influenced by pH suggesting that electrostatic interactions are important only during cluster formation. Cleavage of disulfide bonds and methylation of carboxyl groups in PLP greatly reduced the aggregating activity, indicating that the process is dependent on the protein's conformation. Unexpectedly, the proteolipid DM-20 was also effective at inducing the clustering of neutral lipid vesicles. In contrast, three protein fractions comprising the naturally-occurring PLP fragments 1-107/112, 113/125-276 and 129/131-276, bearing different net charges, displayed a much lower activity. In addition, trypsin digestion of PLP resulted in a progressive decrease in the protein's ability to induce vesicle aggregation which coincided with the disappearance of the full-length molecule. Together, these results suggest that even large PLP fragments cannot fulfill the adhesive function of the intact protein.

Mutant Plp/DM20 cannot be processed to secrete PLP-related oligodendrocyte differentiation/survival factor.

Most of the mutations within the PLP gene result in degeneration of oligodendrocytes and this is believed to be caused by intracellular trafficking defects. Previous studies have demonstrated that cells expressing the wild type PLP gene release a factor promoting differentiation/survival of oligodendrocyte and that this factor is the C-terminal portion of the protein itself. In this study we asked how the naturally occurring mutations of the PLP gene (jimpy, jimpy msd, and rumpshaker) affect this activity. We developed a transient expression system for retroviral production and transduction that enabled the expression of mutant PLP/DM20 cDNAs in NIH3T3 cells. None of the NIH3T3 cells producing mutant PLP/DM20s secreted the PLP-related factor that increases the number of oligodendrocytes. Since it has been shown that rumpshaker DM20 can be transported to the cell surface, but its folding is incorrect, absence of secretion of this factor is more heavily attributable to incorrect protein folding than to the defect in the PLP/DM20 trafficking.

Myelin: Delivery by raft.

Recent results suggest that membrane proteins are delivered to the myelin sheath of an oligodendrocyte on rafts with a distinctive lipid composition. The major intrinsic membrane protein of myelin, proteolipid protein, interacts with rafts in oligodendrocytes but not with the different rafts found in other cell types.