Myelination induces axonal hotspots of synaptic vesicle fusion that promote sheath growth

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Function and Mechanism of Myelin Regulation in Alcohol Abuse and Alcoholism

Excessive alcohol use has adverse effects on the central nervous system (CNS) and can lead to alcohol use disorders (AUDs). Recent studies have suggested that myelin reductions may directly contribute to CNS dysfunctions associated with AUDs. Myelin consists of compact lipid membranes wrapped around axons to provide electrical insulation and trophic support. Regulation of myelin is considered as a new form of neural plasticity due to its profound impacts on the computation of neural networks. In this review, the authors first discuss experimental evidence showing how alcohol exposure causes demyelination in different brain regions, often accompanied by deficits in cognition and emotion. Next, they discuss postulated molecular and cellular mechanisms underlying alcohol's impact on myelin. It is clear that more extensive investigations are needed in this important but underexplored research field in order to gain a better understanding of the myelin‐behavior relationship and to develop new treatment strategies for AUDs.     

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.     

High-Frequency Microdomain Ca2+ Transients and Waves during Early Myelin Internode Remodeling

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The Cytoskeletal Components of the Myelin Fraction Are Affected by a Single Intracranial Injection of Apotransferrin in Young Rats

We have previously shown that in rat pups intracranially injected with a single dose of apotransferrin (aTf), there is an early oligodendroglial cell OLGc differentiation. The expression of the mRNAs of myelin basic proteins and of 2,3 cyclic nucleotide 3-phosphodiesterase and the amount of the corresponding proteins, as well as myelin glycolipids and phospholipids, were significantly increased in these animals at 10 and 17 days of age. Microtubules and myelin basic proteins appear to be closely associated in OLGc and it has been shown that the mRNAs of myelin basic proteins are concentrated in the OLGc processes. The aim of this work was to clarify if the accelerated myelination produced by aTf could be linked to changes in certain cytoskeletal elements present in the myelin fraction such as tubulin, actin, and different microtubule-associated proteins (MAPs). A significant increase in the expression of the mRNA of tubulin and actin was observed in the brain of the aTf-treated animals. Several MAPs, particularly MAP 1B and stable tubule only peptide as well as actin and tubulin, were markedly increased in the Triton X-100 insoluble pellet obtained from the myelin fraction of these animals. The changes that we have previously described in the myelin of aTf intracranially injected rats, could be the consequence of its action on the cytoskeletal network of the OLGc. An enlargement of this structure would result in a more efficient and faster movement of the different components that are normally transported to the myelin by the cytoskeleton of this cell.     

Generation of an OMgp allelic series in mice

The very limited ability to regenerate axons after injury in the mature mammalian central nervous system (CNS) has been partly attributed to the growth restrictive nature of CNS myelin. Oligodendrocyte myelin glycoprotein (OMgp) was identified as a major myelin‐derived inhibitor of axon growth. However, its role in axon regeneration in vivo is poorly understood. Here we describe the generation and molecular characterization of an OMgp allelic series. With a single gene targeting event and Cre/FLP mediated recombination, we generated an OMgp null allele with a LacZ reporter, one without a reporter gene, and an OMgp conditional allele. This allelic series will aid in the study of OMgp in adult CNS axon regeneration using mouse models of spinal cord injury. The conditional allele will overcome developmental compensation when employed with an inducible Cre, and allows for the study of temporal and tissue/cell type‐specific roles of OMgp in CNS injury‐induced axonal plasticity. genesis 47:751–756, 2009. © 2009 Wiley‐Liss, Inc.     

Release of Proteins from the Surface of Bovine Central Nervous System Myelin by Salts and Phospholipases

Incubation of bovine CNS myelin with phospholipase C from Bacillus cereus under conditions that lead to extensive phospholipid degradation caused 10% of the myelin protein to be released from the membrane. The myelin basic protein (MBP) was a major component of the dissolved protein. Comparable incubations with phospholipase C from Clostridium perfringens, phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, or cabbage phospholipase D removed little MBP. However, concentrations of sodium chloride near 1 M and concentrations of divalent metal ions between 50 and 600 mM released typically 9-12% of the total myelin protein, with MBP again as the predominant component. Repetitive washing with calcium chloride solutions resulted in dissolution of over 90% of the MBP. When myelin was incubated in 1.0 M sodium chloride or 25 mM calcium chloride, the MBP was cleaved largely into two major peptides with apparent molecular weights near 14,000 and 12,000, but with 200 mM or higher concentrations of calcium chloride most of this protein remained intact. With appropriate manipulation of the ionic milieu, it is thus possible to remove most of this extrinsic protein from the myelin surface under relatively mild conditions. The conditions that release the protein suggest that it is held at the membrane surface by ionic interactions.     

Temporal Profile of Astrocytes and Changes of Oligodendrocyte-Based Myelin Following Middle Cerebral Artery Occlusion in Diabetic and Non-diabetic Rats

The long-term impacts of cerebral ischemia and diabetic ischemia on astrocytes and oligodendrocytes have not been defined. The objective of this study is to define profile of astrocyte and changes of myelin in diabetic and non-diabetic rats subjected to focal ischemia.Focal cerebral ischemia of 30-min duration was induced in streptozotocin-induced diabetic and vehicle-injected normoglycemic rats. The brains were harvested for immunohistochemistry of glial fibrillary acidic protein (GFAP) and 2'', 3''-cyclic nucleotide 3''-phosphodiesterase (CNPase) at various reperfusion endpoints ranging from 30 min up to 28 days. The results showed that activate astrocytes were observed after 30 min and peaked at 3 h to 1 day after reperfusion in ischemic penumbra, and peaked at 7 days of reperfusion in ischemic core. Diabetes inhibited the activation of astrocytes in ischemic hemisphere. Demyelination occurred after 30 min of reperfusion in ischemic core and peaked at 1 day. Diabetes caused more severe demyelination compared with non-diabetic rats. Remyelination started at 7 days and completed at 14 and 28 days in ischemic region. Diabetes inhibited the remyelination processes. It is concluded that ischemia activates astrocytes and induces demyelination. Diabetes inhibits the activation of astrocytes, exacerbates the demyelination and delays the remyelination processes. These may contribute to the detrimental effects of hyperglycemia on ischemic brain damage.     

iPSC-derived myelinoids to study myelin biology of humans

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The Constitutive Heat Shock Protein-70 Is Required for Optimal Expression of Myelin Basic Protein During Differentiation of Oligodendrocytes

To further elucidate the role of the constitutive heat shock protein-70 (HSC70) as a chaperone for the synthesis of myelin basic protein (MBP), HSC70 content was decreased in oligodendrocyte precursor cells prior to MBP expression either by transfection with an antisense oligonucleotide specific for HSC70, or by exposure to low levels of quercetin, a bioflavonoid known to decrease synthesis of HSC70. As these cells underwent differentiation in vitro, antisense treatment decreased HSC70 levels to 66% of controls. At the same time, a sharp induction resulted in the stress-inducible heat shock protein-70 (HSP70). Levels of two other stress proteins increased as well, namely, the 25-kDa heat shock protein (HSP25) and the 78-kDa glucose regulated protein (GRP78). MBP synthesis proceeded over a normal time course, but at only 50% of control values. As HSC70 content returned to normal, MBP synthesis was also restored to normal levels. Quercetin reduced the expression of HSC70 to an even greater extent than transfection, and prevented the induction of HSP70. In contrast to antisense-treated cells, MBP synthesis was essentially blocked in quercetin-treated cells even though levels of HSP25 and GRP78 increased. Taken together, these observations (a) indicate that HSP70 partially compensates for decreased chaperoning of nascent MBP by HSC70 (HSC70 and HSP70 are closely related and perform similar functions); (b) preclude the involvement of HSP25 and GRP78 in MBP synthesis; and (c) emphasize the requirement of HSC70 for optimal synthesis of MBP.     

Neuronal loss and microgliosis are restricted to the core of Aβ deposits in mouse models of Alzheimer's disease

Amyloid‐β (Aβ) deposits, pathologic tau, and neurodegeneration are major pathological hallmarks of Alzheimer''s disease (AD). The relationship between neuronal loss and Aβ deposits is one of the fundamental questions in the pathogenesis of AD. However, this relationship is controversial. One main reason for the conflicting results may be the confounding effects of pathologic tau, which often coexists with Aβ deposits in the brains of AD patients. To clarify the relationship between neuronal loss and Aβ deposits, mouse models of AD, which develop abundant Aβ deposits in the aged brain without pathologic tau, were used to examine the co‐localization of NeuN‐positive neurons, NF‐H‐positive axons, MBP‐positive myelin sheaths, and Aβ deposits. Neuronal loss, as measured by decreased staining of the neuronal cell body, axon, and myelin sheath, as well as the IBA‐1‐positive microglia, was significantly increased in the core area of cerebral Aβ deposits, but not in adjacent areas. Furthermore, neuronal loss in the core area of cerebral Aβ deposits was correlated with Aβ deposit size. These results clearly indicate that neuronal loss is restricted to the core of Aβ deposits, and this restricted loss probably occurs because the Aβ deposit attracts microglia, which cluster in the core area where Aβ toxicity and neuroinflammation toxicity are restrained. These findings may contribute to our understanding of the relationship between neuronal loss and Aβ deposits in the absence of pathologic tau.     

Neuronal Galectin‐4 is required for axon growth and for the organization of axonal membrane L1 delivery and clustering

Axon membrane glycoproteins are essential for neuronal differentiation, although the mechanisms underlying their polarized sorting and organization are poorly understood. We describe here that galectin‐4 (Gal‐4), a lectin highly expressed in gastrointestinal tissues and involved in epithelial glycoprotein transport, is expressed by hippocampal and cortical neurons where it is sorted to discrete segments of the axonal membrane in a microtubule‐ and sulfatide‐dependent manner. Gal‐4 knockdown retards axon growth, an effect that can be rescued by recombinant Gal‐4 addition. This Gal‐4 reduction, as inhibition of sulfatide synthesis does, lowers the presence and clustered organization of axon growth‐promoting molecule NCAM L1 at the axon membrane. Furthermore, we find that Gal‐4 interacts with L1 by specifically binding to LacNAc branch ends of L1 N‐glycans. Impairing the maturation of these N‐glycans precludes Gal‐4/L1 association resulting in a failure of L1 membrane cluster organization. In all, Gal‐4 sorts to axon plasma membrane segments by binding to sulfatide‐containing microtubule‐associated carriers and being bivalent, it organizes the transport of L1, and likely other axonal glycoproteins, by attaching them to the carriers through their LacNAc termini. This mechanism would underlie L1 functional organization on the plasma membrane, required for proper axon growth.     

Oligodendroglial ring finger protein Rnf43 is an essential injury-specific regulator of oligodendrocyte maturation

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Axonal transport of microtubules: the long and short of it

Recent studies on cultured neurons have demonstrated that microtubules are transported down the axon in the form of short polymers. The transport of these microtubules is bidirectional, intermittent, asynchronous, and occurs at the fast rate of known motors. The majority of the microtubule mass in the axon exists in the form of longer immobile microtubules. We have proposed a model called 'cut and run', in which the longer microtubules are mobilized by enzymes that sever them into shorter mobile polymers. In this view, the molecular motors that transport microtubules are not selective for short microtubules but rather impinge upon microtubules irrespective of their length. In the case of the longer microtubules, these motor-driven forces do not transport the microtubules in a rapid and concerted fashion but presumably affect them nonetheless. Here, we discuss the mechanisms by which the short microtubules are transported and suggest possibilities for how analogous mechanisms may align and organize the longer microtubules and functionally integrate them with each other and with the actin cytoskeleton.     

Myelin suppresses axon regeneration by PIR-B/SHP-mediated inhibition of Trk activity

Paired immunoglobulin-like receptor B (PIR-B) partially mediates the regeneration-inhibiting effects of the myelin-derived protein Nogo, myelin-associated glycoprotein (MAG), and oligodendrocyte-myelin glycoprotein (OMgp). In this study, we report that inhibition of the PIR-B signaling cascades in neurons enhances axon regeneration in the central nervous system (CNS). Binding of MAG to PIR-B led to the association of PIR-B with tropomyosin receptor kinase (Trk) neurotrophin receptors. Src homology 2-containing protein tyrosine phosphatase (SHP)-1 and SHP-2, which were recruited to PIR-B upon MAG binding, functioned as Trk tyrosine phosphatases. Further, SHP-1 and SHP-2 inhibition reduced MAG-induced dephosphorylation of Trk receptors and abolished the inhibitory effect of MAG on neurite growth. Thus, PIR-B associated with Trk to downregulate basal and neurotrophin-regulated Trk activity through SHP-1/2 in neurons. Moreover, in vivo transfection of small interfering RNA (siRNA) for SHP-1 or SHP-2 induced axonal regeneration after optic nerve injury in mice. Our results thus identify a new molecular target to enhance regeneration of the injured CNS.     

Transgenic mice expressing a dual, CRE-inducible reporter for the analysis of axon guidance and synaptogenesis

Improved and modular tools are needed for the neuroanatomical dissection of CNS axonal tracts, and to study the cell-intrinsic and cell-extrinsic cues that govern their assembly and plasticity. Here we describe a general purpose transgenic tracer that can be used to visualize axonal tracts and synaptic terminals in any region of the embryonic neural tube or postnatal CNS, on any wild type or mutant genetic background. The construct permits CRE-inducible expression of a dicistronic axonal marker encoding two surface reporter proteins: a farnesylated GFP and the human Placental Alkaline Phosphatase (PLAP). Both proteins localize alongside the neuronal surface, permitting the concomitant detection of cell body, neurites, and presynaptic and postsynaptic sites in the same neuron. This provides a CRE-inducible dual system for imaging neural circuits in vivo, and to study their assembly and remodeling in cultured neurons, neural stem cells, and tissue explants derived from the reporter line. Unlike existing lines, this reporter does not encode a ubiquitously expressed, floxable LacZ gene, permitting the simultaneous analysis of beta galactosidase activity in mutant lines.     

Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms

The only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are targeted to distinct subcellular locations, which is governed by the presence or absence of exon-II, although their functional expression is often less clear. Here, we investigated the role of exon-II-containing MBP isoforms and their link with cell proliferation. Live-cell imaging and FRAP analysis revealed a dynamic nucleocytoplasmic translocation of the exon-II-containing postnatal 21.5-kDa MBP isoform upon mitogenic modulation. Its nuclear export was blocked upon treatment with leptomycin B, an inhibitor of nuclear protein export. Next to the postnatal MBP isoforms, embryonic exon-II-containing MBP (e-MBP) is expressed in primary (immature) oligodendrocytes. The e-MBP isoform is exclusively present in OLN-93 cells, a rat-derived oligodendrocyte progenitor cell line, and interestingly, also in several non-CNS cell lines. As seen for postnatal MBPs, a similar nucleocytoplasmic translocation upon mitogenic modulation was observed for e-MBP. Thus, upon serum deprivation, e-MBP was excluded from the nucleus, whereas re-addition of serum re-established its nuclear localization, with a concomitant increase in proliferation. Knockdown of MBP by shRNA confirmed a role for e-MBP in OLN-93 proliferation, whereas the absence of e-MBP similarly reduced the proliferative capacity of non-CNS cell lines. Thus, exon-II-containing MBP isoforms may regulate cell proliferation via a mechanism that relies on their dynamic nuclear import and export, which is not restricted to the oligodendrocyte lineage.     

On the biogenesis of myelin membranes: Sorting, trafficking and cell polarity

In the central nervous system, a multilayered membrane layer known as the myelin sheath enwraps axons, and is required for optimal saltatory signal conductance. The sheath develops from membrane processes that extend from the plasma membrane of oligodendrocytes and displays a unique lipid and protein composition. Myelin biogenesis is carefully regulated, and multiple transport pathways involving a variety of endosomal compartments are involved. Here we briefly summarize how the major myelin proteins proteolipid protein and myelin basic protein reach the sheath, and highlight potential mechanisms involved, including the role of myelin specific lipids and cell polarity related transport pathways.     

Novel inhibitors of leukocyte transendothelial migration

Leukocyte transendothelial migration is one of the most important step in launching an inflammatory immune response and chronic inflammation can lead to devastating diseases. Leukocyte migration inhibitors are considered as promising and potentially effective therapeutic agents to treat inflammatory and auto-immune disorders. In this study, based on previous trioxotetrahydropyrimidin based integrin inhibitors that suboptimally blocked leukocyte adhesion, twelve molecules with a modified scaffold were designed, synthesized, and tested in vitro for their capacity to block the transendothelial migration of immune cells. One of the molecules, namely, methyl 4-((2-(tert-butyl)-6-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) methyl) phenoxy) methyl) benzoate, (compound 12), completely blocked leukocyte transendothelial migration, without any toxic effects on immune or endothelial cells (IC₅₀ = 2.4 µM). In vivo, compound 12 exhibited significant therapeutic effects in inflammatory bowel disease (IBD)/Crohn’s disease, multiple sclerosis, fatty liver disease, and rheumatoid arthritis models. A detailed acute and chronic toxicity profile of the lead compound in vivo did not reveal any toxic effects. Such a type of molecule might therefore provide a unique starting point for designing a novel class of leukocyte transmigration blocking agents with broad therapeutic applications in inflammatory and auto-immune pathologies.