LINGO-1 and its role in CNS repair

LINGO-1 is selectively expressed in the CNS on both oligodendrocyte precursor cells (OPCs) and neurons. Its expression is developmentally regulated in the normal CNS, as well as up-regulated in human or rat models of neuropathologies. LINGO-1 functions as a negative regulator of oligodendrocyte differentiation and myelination, neuronal survival and axonal regeneration. Across diverse animal CNS disease models, targeted LINGO-1 inhibition was found to promote neuron and oligodendrocyte survival, axon regeneration, oligodendrocyte differentiation, remyelination and improved functional recovery. The targeted inhibition of LINGO-1 therefore presents a novel therapeutic approach for the treatment of neurological diseases.     

Production of a PEGylated Fab' of the anti-LINGO-1 Li33 antibody and assessment of its biochemical and functional properties in vitro and in a rat model of remyelination

The use of LINGO-1 antagonists to promote repair of damaged myelin is an emerging therapeutic opportunity for treatment of CNS diseases caused by demyelination such as multiple sclerosis. The Li33 anti-LINGO-1 antibody is a potent inducer of myelination in vitro and in vivo, but aggregation issues prevented the engineering of an optimal development candidate. PEGylated Li33 Fab' is one of several versions of the Li33 antibody that is being investigated in an attempt to identify the most favorable anti-LINGO-1 antibody design. For targeted PEGylation, a Li33 Fab' construct was engineered with a single unpaired cysteine in the heavy-chain hinge sequence. The Fab' was expressed in CHO cells, purified, and PEGylated with 20 kDa methoxy-poly(ethylene glycol) maleimide using a reaction strategy optimized to improve the yield of the PEG-Fab'. Biochemical analysis of the Li33 PEG-Fab' verified the selectivity of the PEGylation reaction. The in vitro and in vivo attributes of the PEG-Fab' were benchmarked against a Li33 full antibody. Both the Li33 PEG-Fab' and intact antibody bound LINGO-1 with nanomolar affinity, promoted myelination in an in vitro signaling assay, and promoted the repair of damaged myelin in the rat lysolecithin model. These studies extend our understanding of the biological activity of the Li33 mAb and validate the use of an anti-LINGO-1 PEG-Fab' for treatment of CNS diseases caused by demyelination.     

LINGO-1-Fc蛋白对低钾诱导小脑颗粒神经元凋亡的保护作用

髓鞘抑制因子Nogo-A、MAG和OMgp通过共同的受体信号复合物NgR/p75NTR(或者TROY)发挥对中枢神经纤维再生的抑制作用.新近克隆的跨膜蛋白LINGO-1是该信号途径的另一个重要组成成分和调节分子.LINGO-1特异表达于中枢神经系统,神经元上的LINGO-1被证明参与调节中枢神经再生的抑制信号,而少突胶质细胞表达的LINGO-1分子参与负调节少突胶质细胞的髓鞘化过程.为探讨LINGO-1分子在神经元凋亡过程中的作用,利用包含LINGO-1分子胞外段LRR和IgC2结构域的Fc融合蛋白作为功能性拮抗剂,研究LINGO-1对低钾诱导的小脑颗粒神经元凋亡的保护作用.利用成熟的Hoechst标记凋亡细胞的方法,观察到经LINGO-1-Fc蛋白预处理2h能够显著阻止小脑颗粒神经元的凋亡.仅包括LRR结构域的GST-LINGO-1与LINGO-1-Fc蛋白,虽同样具有与颗粒神经元的结合活性,但是GST-LINGO-1不能有效地阻止低钾诱导的细胞凋亡.这些结果提示,LINGO-1-Fc蛋白能够阻止低钾诱导的小脑颗粒神经元凋亡,并且这种作用可能是IgC2结构域依赖的.     

LINGO-1, a transmembrane signaling protein, inhibits oligodendrocyte differentiation and myelination through intercellular self-interactions

Overcoming remyelination failure is a major goal of new therapies for demyelinating diseases like multiple sclerosis. LINGO-1, a key negative regulator of myelination, is a transmembrane signaling protein expressed in both neurons and oligodendrocytes. In neurons, LINGO-1 is an integral component of the Nogo receptor complex, which inhibits axonal growth via RhoA. Because the only ligand-binding subunit of this complex, the Nogo receptor, is absent in oligodendrocytes, the extracellular signals that inhibit myelination through a LINGO-1-mediated mechanism are unknown. Here we show that LINGO-1 inhibits oligodendrocyte terminal differentiation through intercellular interactions and is capable of a self-association in trans. Consistent with previous reports, overexpression of full-length LINGO-1 inhibited differentiation of oligodendrocyte precursor cells (OPCs). Unexpectedly, treatment with a soluble recombinant LINGO-1 ectodomain also had an inhibitory effect on OPCs and decreased myelinated axonal segments in cocultures with neurons from dorsal root ganglia. We demonstrated LINGO-1-mediated inhibition of OPCs through intercellular signaling by using a surface-bound LINGO-1 construct expressed ectopically in astrocytes. Further investigation showed that the soluble LINGO-1 ectodomain can interact with itself in trans by binding to CHO cells expressing full-length LINGO-1. Finally, we observed that soluble LINGO-1 could activate RhoA in OPCs. We propose that LINGO-1 acts as both a ligand and a receptor and that the mechanism by which it negatively regulates OPC differentiation and myelination is mediated by a homophilic intercellular interaction. Disruption of this protein-protein interaction could lead to a decrease of LINGO-1 inhibition and an increase in myelination.     

Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin

Cholesterol availability is rate-limiting for myelination, and prior studies have established the importance of cholesterol synthesis by oligodendrocytes for normal CNS myelination. However, the contribution of cholesterol uptake through the endocytic pathway has not been fully explored. To address this question, we used mice with a conditional null allele of the Npc1 gene, which encodes a transmembrane protein critical for mobilizing cholesterol from the endolysosomal system. Loss of function mutations in the human NPC1 gene cause Niemann-Pick type C disease, a childhood-onset neurodegenerative disorder in which intracellular lipid accumulation, abnormally swollen axons, and neuron loss underlie the occurrence of early death. Both NPC patients and Npc1 null mice exhibit myelin defects indicative of dysmyelination, although the mechanisms underlying this defect are incompletely understood. Here we use temporal and cell-type-specific gene deletion in order to define effects on CNS myelination. Our results unexpectedly show that deletion of Npc1 in neurons alone leads to an arrest of oligodendrocyte maturation and to subsequent failure of myelin formation. This defect is associated with decreased activation of Fyn kinase, an integrator of axon-glial signals that normally promotes myelination. Furthermore, we show that deletion of Npc1 in oligodendrocytes results in delayed myelination at early postnatal days. Aged, oligodendocyte-specific null mutants also exhibit late stage loss of myelin proteins, followed by secondary Purkinje neuron degeneration. These data demonstrate that lipid uptake and intracellular transport by neurons and oligodendrocytes through an Npc1-dependent pathway is required for both the formation and maintenance of CNS myelin.     

Cdk2 loss accelerates precursor differentiation and remyelination in the adult central nervous system

The specific functions of intrinsic regulators of oligodendrocyte progenitor cell (OPC) division are poorly understood. Type 2 cyclin-dependent kinase (Cdk2) controls cell cycle progression of OPCs, but whether it acts during myelination and repair of demyelinating lesions remains unexplored. Here, we took advantage of a viable Cdk2(-/-) mutant mouse to investigate the function of this cell cycle regulator in OPC proliferation and differentiation in normal and pathological conditions. During central nervous system (CNS) development, Cdk2 loss does not affect OPC cell cycle, oligodendrocyte cell numbers, or myelination. However, in response to CNS demyelination, it clearly alters adult OPC renewal, cell cycle exit, and differentiation. Importantly, Cdk2 loss accelerates CNS remyelination of demyelinated axons. Thus, Cdk2 is dispensable for myelination but is important for adult OPC renewal, and could be one of the underlying mechanisms that drive adult progenitors to differentiate and thus regenerate myelin.     

AMIGO3 Is an NgR1/p75 Co-Receptor Signalling Axon Growth Inhibition in the Acute Phase of Adult Central Nervous System Injury

Axon regeneration in the injured adult CNS is reportedly inhibited by myelin-derived inhibitory molecules, after binding to a receptor complex comprised of the Nogo-66 receptor (NgR1) and two transmembrane co-receptors p75/TROY and LINGO-1. However, the post-injury expression pattern for LINGO-1 is inconsistent with its proposed function. We demonstrated that AMIGO3 levels were significantly higher acutely than those of LINGO-1 in dorsal column lesions and reduced in models of dorsal root ganglion neuron (DRGN) axon regeneration. Similarly, AMIGO3 levels were raised in the retina immediately after optic nerve crush, whilst levels were suppressed in regenerating optic nerves, induced by intravitreal peripheral nerve implantation. AMIGO3 interacted functionally with NgR1-p75/TROY in non-neuronal cells and in brain lysates, mediating RhoA activation in response to CNS myelin. Knockdown of AMIGO3 in myelin-inhibited adult primary DRG and retinal cultures promoted disinhibited neurite growth when cells were stimulated with appropriate neurotrophic factors. These findings demonstrate that AMIGO3 substitutes for LINGO-1 in the NgR1-p75/TROY inhibitory signalling complex and suggests that the NgR1-p75/TROY-AMIGO3 receptor complex mediates myelin-induced inhibition of axon growth acutely in the CNS. Thus, antagonizing AMIGO3 rather than LINGO-1 immediately after CNS injury is likely to be a more effective therapeutic strategy for promoting CNS axon regeneration when combined with neurotrophic factor administration.     

A TNF receptor family member, TROY, is a coreceptor with Nogo receptor in mediating the inhibitory activity of myelin inhibitors

A major obstacle for successful axon regeneration in the adult central nervous system (CNS) arises from inhibitory molecules in CNS myelin, which signal through a common receptor complex on neurons consisting of the ligand-binding Nogo-66 receptor (NgR) and two transmembrane coreceptors, p75 and LINGO-1. However, p75 expression is only detectable in subpopulations of mature neurons, raising the question of how these inhibitory signals are transduced in neurons lacking p75. In this study, we demonstrate that TROY (also known as TAJ), a TNF receptor family member selectively expressed in the adult nervous system, can form a functional receptor complex with NgR and LINGO-1 to mediate cellular responses to myelin inhibitors. Also, both overexpressing a dominant-negative TROY or presence of a soluble TROY protein can efficiently block neuronal response to myelin inhibitors. Our results implicate TROY in mediating myelin inhibition, offering new insights into the molecular mechanisms of regeneration failure in the adult nervous system.     

Identification of dystroglycan as a second laminin receptor in oligodendrocytes, with a role in myelination

Developmental abnormalities of myelination are observed in the brains of laminin-deficient humans and mice. The mechanisms by which these defects occur remain unknown. It has been proposed that, given their central role in mediating extracellular matrix (ECM) interactions, integrin receptors are likely to be involved. However, it is a non-integrin ECM receptor, dystroglycan, that provides the key linkage between the dystrophin-glycoprotein complex (DGC) and laminin in skeletal muscle basal lamina, such that disruption of this bridge results in muscular dystrophy. In addition, the loss of dystroglycan from Schwann cells causes myelin instability and disorganization of the nodes of Ranvier. To date, it is unknown whether dystroglycan plays a role during central nervous system (CNS) myelination. Here, we report that the myelinating glia of the CNS, oligodendrocytes, express and use dystroglycan receptors to regulate myelin formation. In the absence of normal dystroglycan expression, primary oligodendrocytes showed substantial deficits in their ability to differentiate and to produce normal levels of myelin-specific proteins. After blocking the function of dystroglycan receptors, oligodendrocytes failed both to produce complex myelin membrane sheets and to initiate myelinating segments when co-cultured with dorsal root ganglion neurons. By contrast, enhanced oligodendrocyte survival in response to the ECM, in conjunction with growth factors, was dependent on interactions with beta-1 integrins and did not require dystroglycan. Together, these results indicate that laminins are likely to regulate CNS myelination by interacting with both integrin receptors and dystroglycan receptors, and that oligodendrocyte dystroglycan receptors may have a specific role in regulating terminal stages of myelination, such as myelin membrane production, growth, or stability.     

Death receptor 6 negatively regulates oligodendrocyte survival, maturation and myelination

Survival and differentiation of oligodendrocytes are important for the myelination of central nervous system (CNS) axons during development and crucial for myelin repair in CNS demyelinating diseases such as multiple sclerosis. Here we show that death receptor 6 (DR6) is a negative regulator of oligodendrocyte maturation. DR6 is expressed strongly in immature oligodendrocytes and weakly in mature myelin basic protein (MBP)-positive oligodendrocytes. Overexpression of DR6 in oligodendrocytes leads to caspase 3 (casp3) activation and cell death. Attenuation of DR6 function leads to enhanced oligodendrocyte maturation, myelination and downregulation of casp3. Treatment with a DR6 antagonist antibody promotes remyelination in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis (EAE) models. Consistent with the DR6 antagoinst antibody studies, DR6-null mice show enhanced remyelination in both demyelination models. These studies reveal a pivotal role for DR6 signaling in immature oligodendrocyte maturation and myelination that may provide new therapeutic avenues for the treatment of demyelination disorders such as multiple sclerosis.     

TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration

Myelin-associated inhibitory factors (MAIFs) are inhibitors of CNS axonal regeneration following injury. The Nogo receptor complex, composed of the Nogo-66 receptor 1 (NgR1), neurotrophin p75 receptor (p75), and LINGO-1, represses axon regeneration upon binding to these myelin components. The limited expression of p75 to certain types of neurons and its temporal expression during development prompted speculation that other receptors are involved in the NgR1 complex. Here, we show that an orphan receptor in the TNF family called TAJ, broadly expressed in postnatal and adult neurons, binds to NgR1 and can replace p75 in the p75/NgR1/LINGO-1 complex to activate RhoA in the presence of myelin inhibitors. In vitro exogenously added TAJ reversed neurite outgrowth caused by MAIFs. Neurons from Taj-deficient mice were more resistant to the suppressive action of the myelin inhibitors. Given the limited expression of p75, the discovery of TAJ function is an important step for understanding the regulation of axonal regeneration.     

The temporal progression of the myelination defect in the taiep rat

The Sprague Dawley myelin mutant, the taiep rat, demonstrates a defect in CNS myelination which worsens with age and which is associated with abnormal accumulations of microtubules in oligodendrocytes. Quantitative and qualitative electron microscopic studies of myelin development and oligodendrocyte morphology were used to describe the temporal development of the defect in this mutant, in three regions of the CNS. The results indicate that the time of onset of myelination is similar in mutant and control rats, however the amount of myelin formed is reduced in the mutant, compared to controls, and there is a loss of myelin from the taiep CNS as the animals age. Thus the myelination defect in taiep has features of both hypomyelination and demyelination. Oligodendrocyte microtubule abnormalities were noted in each region of the taiep CNS at the time of onset of myelination. The earliest changes seen were close associations of oligodendrocyte microtubules with endoplasmic reticulum, with marked accumulations of microtubules filling the cytoplasm of oligodendrocytes from older taiep rats. These findings suggest that the microtubule abnormality in the taiep mutant inhibits both the initial formation and the long-term maintenance of myelin by the oligodendrocyte. In addition, there is also evidence to suggest that although the microtubule abnormality is present in oligodendrocytes throughout the taiep CNS, it results in a more marked defect in the myelination of axons of small diameter.     

Neuregulin-1/ErbB signaling serves distinct functions in myelination of the peripheral and central nervous system

Understanding the control of myelin formation by oligodendrocytes is essential for treating demyelinating diseases. Neuregulin-1 (NRG1) type III, an EGF-like growth factor, is essential for myelination in the PNS. It is thus thought that NRG1/ErbB signaling also regulates CNS myelination, a view suggested by in vitro studies and the overexpression of dominant-negative ErbB receptors. To directly test this hypothesis, we generated a series of conditional null mutants that completely lack NRG1 beginning at different stages of neural development. Unexpectedly, these mice assemble normal amounts of myelin. In addition, double mutants lacking oligodendroglial ErbB3 and ErbB4 become myelinated in the absence of any stimulation by neuregulins. In contrast, a significant hypermyelination is achieved by transgenic overexpression of NRG1 type I or NRG1 type III. Thus, NRG1/ErbB signaling is markedly different between Schwann cells and oligodendrocytes that have evolved an NRG/ErbB-independent mechanism of myelination control.     

The cellular and molecular events of central nervous system remyelination

Central nervous system (CNS)* regeneration is a subject of great interest, particularly in diseases causing a dramatic loss of neurons. However, some CNS diseases do not affect neurons but damage other cells, such as the myelin-forming cells--called oligodendrocytes--which are also crucial to the harmonious function of the nervous system. Diseases in which oligodendrocytes and myelin are attacked can cause devastating neurological dysfunction which is sometimes followed by recovery and myelin repair or remyelination. The question of the regeneration potential of oligodendrocytes in experimental and human demyelinating diseases such as multiple sclerosis has been debated for a long time. Present evidence suggests that oligodendrocyte precursor cells persist in the adult CNS and that oligodendrocyte regeneration can occur but may be limited by ongoing disease processes. Here we will briefly review recent advances which have broadened our understanding of the cellular and molecular events of CNS remyelination.     

Protein Tyrosine Phosphatase Receptor Type Z Negatively Regulates Oligodendrocyte Differentiation and Myelination

Background

Fyn tyrosine kinase-mediated down-regulation of Rho activity through activation of p190RhoGAP is crucial for oligodendrocyte differentiation and myelination. Therefore, the loss of function of its counterpart protein tyrosine phosphatase (PTP) may enhance myelination during development and remyelination in demyelinating diseases. To test this hypothesis, we investigated whether Ptprz, a receptor-like PTP (RPTP) expressed abuntantly in oligodendrocyte lineage cells, is involved in this process, because we recently revealed that p190RhoGAP is a physiological substrate for Ptprz.

Methodology/Principal Findings

We found an early onset of the expression of myelin basic protein (MBP), a major protein of the myelin sheath, and early initiation of myelination in vivo during development of the Ptprz-deficient mouse, as compared with the wild-type. In addition, oligodendrocytes appeared earlier in primary cultures from Ptprz-deficient mice than wild-type mice. Furthermore, adult Ptprz-deficient mice were less susceptible to experimental autoimmune encephalomyelitis (EAE) induced by active immunization with myelin/oligodendrocyte glycoprotein (MOG) peptide than were wild-type mice. After EAE was induced, the tyrosine phosphorylation of p190RhoGAP increased significantly, and the EAE-induced loss of MBP was markedly suppressed in the white matter of the spinal cord in Ptprz-deficient mice. Here, the number of T-cells and macrophages/microglia infiltrating into the spinal cord did not differ between the two genotypes after MOG immunization. All these findings strongly support the validity of our hypothesis.

Conclusions/Significance

Ptprz plays a negative role in oligodendrocyte differentiation in early central nervous system (CNS) development and remyelination in demyelinating CNS diseases, through the dephosphorylation of substrates such as p190RhoGAP.     

The Polarity Protein Scribble Regulates Myelination and Remyelination in the Central Nervous System

The development and regeneration of myelin by oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), requires profound changes in cell shape that lead to myelin sheath initiation and formation. Here, we demonstrate a requirement for the basal polarity complex protein Scribble in CNS myelination and remyelination. Scribble is expressed throughout oligodendroglial development and is up-regulated in mature oligodendrocytes where it is localised to both developing and mature CNS myelin sheaths. Knockdown of Scribble expression in cultured oligodendroglia results in disrupted morphology and myelination initiation. When Scribble expression is conditionally eliminated in the myelinating glia of transgenic mice, myelin initiation in CNS is disrupted, both during development and following focal demyelination, and longitudinal extension of the myelin sheath is disrupted. At later stages of myelination, Scribble acts to negatively regulate myelin thickness whilst suppressing the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAP) kinase pathway, and localises to non-compact myelin flanking the node of Ranvier where it is required for paranodal axo-glial adhesion. These findings demonstrate an essential role for the evolutionarily-conserved regulators of intracellular polarity in myelination and remyelination.     

Unconventional Myosin ID is Involved in Remyelination After Cuprizone-Induced Demyelination

Myelin, which is a multilamellar structure that sheathes the axon, is essential for normal neuronal function. In the central nervous system (CNS), myelin is produced by oligodendrocytes (OLs), which wrap their plasma membrane around axons. The dynamic membrane trafficking system, which relies on motor proteins, is required for myelin formation and maintenance. Previously, we reported that myosin ID (Myo1d) is distributed in rat CNS myelin and is especially enriched in the outer and inner cytoplasm-containing loops. Further, small interfering RNA (siRNA) treatment highlighted the involvement of Myo1d in the formation and maintenance of myelin in cultured OLs. Myo1d is one of the unconventional myosins, which may contribute to membrane dynamics, either in the wrapping process or transport of myelin membrane proteins during myelination. However, the function of Myo1d in myelin formation in vivo remains unclear. In the current study, to clarify the function of Myo1d in vivo, we surgically injected siRNA in the corpus callosum of a cuprizone-treated demyelination mouse model via stereotaxy. Knockdown of Myo1d expression in vivo decreased the intensities of myelin basic protein and myelin proteolipid protein immunofluorescence staining. However, neural/glial antigen 2-positive signals and adenomatous polyposis coli (APC/CC1)-positive cell numbers were unchanged by siRNA treatment. Furthermore, Myo1d knockdown treatment increased pro-inflammatory microglia and astrocytes during remyelination. In contrast, anti-inflammatory microglia were decreased. The percentage of caspase 3-positive cells in total CC1-positive OLs were also increased by Myo1d knockdown. These results indicated that Myo1d plays an important role during the regeneration process after demyelination.