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.     

LINGO-1 antagonist promotes spinal cord remyelination and axonal integrity in MOG-induced experimental autoimmune encephalomyelitis

Demyelinating diseases, such as multiple sclerosis, are characterized by the loss of the myelin sheath around neurons, owing to inflammation and gliosis in the central nervous system (CNS). Current treatments therefore target anti-inflammatory mechanisms to impede or slow disease progression. The identification of a means to enhance axon myelination would present new therapeutic approaches to inhibit and possibly reverse disease progression. Previously, LRR and Ig domain-containing, Nogo receptor-interacting protein (LINGO-1) has been identified as an in vitro and in vivo negative regulator of oligodendrocyte differentiation and myelination. Here we show that loss of LINGO-1 function by Lingo1 gene knockout or by treatment with an antibody antagonist of LINGO-1 function leads to functional recovery from experimental autoimmune encephalomyelitis. This is reflected biologically by improved axonal integrity, as confirmed by magnetic resonance diffusion tensor imaging, and by newly formed myelin sheaths, as determined by electron microscopy. Antagonism of LINGO-1 or its pathway is therefore a promising approach for the treatment of demyelinating diseases of the CNS.     

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.     

Inactivation of Glycogen Synthase Kinase-3β and Up-regulation of LINGO-1 are Involved in LINGO-1 Antagonist Regulated Survival of Cerebellar Granular Neurons

LINGO-1 has been critically implicated in the central regulation of CNS axon regeneration and oligodendrocyte maturation. We have recently demonstrated that pretreatment with LINGO-1 antagonist (LINGO-1-Fc) inhibited low potassium-induced cerebellar granular neurons (CGNs) apoptosis. In the present study, we examined the neuroprotective mechanism of LINGO-1-Fc by Western blot and in situ GST pull-down assay. CGN cultures were preincubated in medium with LINGO-1-Fc or control protein at the concentration of 10 mug/ml for 2 h and then switched to low potassium medium in the presence of corresponding proteins. Cultures were harvested at indicated time intervals for successive analysis. Several apoptosis-associated signaling factors, GSK-3beta, ERK1/2, and Rho GTPases, were observed to be activated in response to potassium deprivation and the activation/dephosphorylation of GSK-3beta was suppressed by LINGO-1-Fc pretreatment compared with control group. Besides, the endogenous LINGO-1 expression level of CGN cultures was augmented by low potassium stimuli and restrained by LINGO-1 antagonist treatment. Although the protein level of p75(NTR) and Nogo-A were down-regulated in different patterns during apoptosis, neither of them was affected by LINGO-1-Fc application. Taken together, these results suggest a new mechanism of LINGO-1 antagonist regulated neuronal survival involving protein synthesis of LINGO-1 and inactivation of GSK-3 pathway.     

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.     

LINGO-1 Protein Interacts with the p75 Neurotrophin Receptor in Intracellular Membrane Compartments

Axon outgrowth inhibition in response to trauma is thought to be mediated via the binding of myelin-associated inhibitory factors (e.g. Nogo-66, myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and myelin basic protein) to a putative tripartite LINGO-1·p75^NTR·Nogo-66 receptor (NgR) complex at the cell surface. We found that endogenous LINGO-1 expression in neurons in the cortex and cerebellum is intracellular. Mutation or truncation of the highly conserved LINGO-1 C terminus altered this intracellular localization, causing poor intracellular retention and increased plasma membrane expression. p75^NTR associated predominantly with natively expressed LINGO-1 containing immature N-glycans, characteristic of protein that has not completed trans-Golgi-mediated processing, whereas mutant forms of LINGO-1 with enhanced plasma membrane expression did not associate with p75^NTR. Co-immunoprecipitation experiments demonstrated that LINGO-1 and NgR competed for binding to p75^NTR in a manner that is difficult to reconcile with the existence of a LINGO-1·p75^NTR·NgR ternary complex. These findings contradict models postulating functional LINGO-1·p75^NTR·NgR complexes in the plasma membrane.     

Contact with central nervous system myelin inhibits oligodendrocyte progenitor maturation

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated during development through the proliferation and differentiation of a distinct progenitor population. Not all oligodendrocyte progenitors generated during development differentiate, however, and large numbers of oligodendrocyte progenitors are present in the adult CNS, particularly in white matter. These "adult progenitors" can be identified through expression of the NG2 proteoglycan. Adult oligodendrocyte progenitors are thought to develop from the original pool of progenitors and in vitro are capable of differentiating into oligodendrocytes. Why these cells fail to differentiate in the intact CNS is currently unclear. Here we show that contact with CNS myelin inhibits the maturation of immature oligodendrocyte progenitors. The inhibition of oligodendrocyte progenitor maturation is a characteristic of CNS myelin that is not shared by several other membrane preparations including adult and neonatal neural membrane fractions, PNS myelin, or liver. This inhibition is concentration dependent, is reversible, and appears not to be mediated by either myelin basic protein or basic fibroblast growth factor. Myelin-induced inhibition of oligodendrocyte progenitor maturation provides a mechanism to explain the generation of a residual pool of immature oligodendrocyte progenitors in the mature CNS.     

Pelizaeus-Merzbacher disease-associated proteolipid protein 1 inhibits oligodendrocyte precursor cell differentiation via extracellular-signal regulated kinase signaling

Oligodendrocytes (OLs) are myelin-forming glial cells in the central nervous system (CNS) and their dysfunction causes neuropathies such as demyelinating diseases. Proteolipid protein 1 (PLP1) is an oligodendrocyte myelin-rich tetraspan membrane protein and aberration of the plp1 gene is known to be responsible for dysmyelinating Pelizaeus-Merzbacher disease (PMD). Among previously identified gene alternations, multiplication of the plp1 gene causes increased expression of PLP1, resulting in a phenotype with severe dysmyelination in human and also rodent models. Yet little is known about the relationship between increased PLP1 expression and oligodendrocyte precursor cell (OPC) differentiation and the intracellular molecular mechanism. Here we show that expression of PLP1 in OPCs markedly inhibits their differentiation, and that this inhibitory effect is effectively improved by inhibition of extracellular signal-regulated kinase (ERK) activity. Furthermore, in cocultures with dorsal root ganglion (DRG) neurons, ERK inhibition also improves PLP1-induced dysmyelination. Thus, ERK inhibition helps to improve defective OPC differentiation induced by PLP1 expression, suggesting that molecules belonging to ERK signaling cascade may be new PMD therapeutic targets.     

Regenerative medicine in multiple sclerosis: identifying pharmacological targets of adult neural stem cell differentiation

Progressive axonal loss from chronic demyelination in multiple sclerosis (MS) is the key contributor to clinical decline. Failure to regenerate myelin by adult oligodendrocyte precursor cells (OPCs), a widely distributed neural stem cell population in the adult brain, is one of the major causes of axonal degeneration. In order to develop successful therapies to protect the integrity of axons in MS, it is important to identify and understand the key molecular pathways involved in myelin regeneration (remyelination) by adult OPCs. This review highlights recent findings on the critical signaling pathways associated with OPC differentiation following CNS demyelination. We discuss the role of LINGO-1, Notch, Wnt, and retinoid X receptor (RXR) signaling, and how they might be useful pharmacological targets to overcoming remyelination failure in MS.     

Neutralization of LINGO-1 during in vitro differentiation of neural stem cells results in proliferation of immature neurons

Identifying external factors that can be used to control neural stem cells division and their differentiation to neurons, astrocytes and oligodendrocytes is of high scientific and clinical interest. Here we show that the Nogo-66 receptor interacting protein LINGO-1 is a potent regulator of neural stem cell maturation to neurons. LINGO-1 is expressed by cortical neural stem cells from E14 mouse embryos and inhibition of LINGO-1 during the first days of neural stem cell differentiation results in decreased neuronal maturation. Compared to neurons in control cultures, which after 6 days of differentiation have long extending neurites, neurons in cultures treated with anti-LINGO-1 antibodies retain an immature, round phenotype with only very short processes. Furthermore, neutralization of LINGO-1 results in a threefold increase in βIII tubulin-positive cells compared to untreated control cultures. By using BrdU incorporation assays we show that the immature neurons in LINGO-1 neutralized cultures are dividing neuroblasts. In contrast to control cultures, in which no cells were double positive for βIII tubulin and BrdU, 36% of the neurons in cultures treated with anti-LINGO-1 antibodies were proliferating after three days of differentiation. TUNEL assays revealed that the amount of cells going through apoptosis during the early phase of differentiation was significantly decreased in cultures treated with anti-LINGO-1 antibodies compared to untreated control cultures. Taken together, our results demonstrate a novel role for LINGO-1 in neural stem cell differentiation to neurons and suggest a possibility to use LINGO-1 inhibitors to compensate for neuronal cell loss in the injured brain.     

LINGO-1 Interacts with WNK1 to Regulate Nogo-induced Inhibition of Neurite Extension

LINGO-1 is a component of the tripartite receptor complexes, which act as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors and lead to collapse of growth cone and inhibition of neurite extension. Although the function of LINGO-1 has been intensively studied, its downstream signaling remains elusive. In the present study, a novel interaction between LINGO-1 and a serine-threonine kinase WNK1 was identified by yeast two-hybrid screen. The interaction was further validated by fluorescence resonance energy transfer and co-immunoprecipitation, and this interaction was intensified by Nogo66 treatment. Morphological evidences showed that WNK1 and LINGO-1 were co-localized in cortical neurons. Furthermore, either suppressing WNK1 expression by RNA interference or overexpression of WNK1-(123–510) attenuated Nogo66-induced inhibition of neurite extension and inhibited the activation of RhoA. Moreover, WNK1 was identified to interact with Rho-GDI1, and this interaction was attenuated by Nogo66 treatment, further indicating its regulatory effect on RhoA activation. Taken together, our results suggest that WNK1 is a novel signaling molecule involved in regulation of LINGO-1 mediated inhibition of neurite extension.Axons of the adult mammalian central nervous system possess an extremely limited ability to regenerate after injury, largely because of inhibitory components of myelin preventing axon growth (1, 2). Several myelin-associated inhibitors have been identified, including myelin-associated glycoprotein (3–5), chondroitin sulfate proteoglycans (6), oligodendrocyte myelin glycoprotein (7), and Nogo (8–10). Myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and Nogo bind to the Nogo-66 receptor (NgR)3 and exert their actions through a tripartite receptor complex NgR/LINGO-1/p75^NTR (11) or NgR/LINGO-1/TROY (12, 13).LINGO-1 is a transmembrane protein that contains a leucine-rich repeat, an immunoglobulin domain, and a short intracellular tail (11). LINGO-1 functions as an essential component of the NgR complexes that mediate the activity of myelin inhibitors to regulate central nervous system axon growth (11, 14). In neurons, the NgR complexes activate RhoA in the presence of myelin inhibitors, which lead to growth cone collapse and neurite extension inhibition (11). Attenuation of LINGO-1 function is able to overcome the myelin inhibitory activity in the spinal cord that prevents axonal regeneration after lesion in rats (15). Besides, it has been reported that LINGO-1 is also expressed in oligodendrocytes, where it negatively regulates oligodendrocyte differentiation and axon myelination (16). Inhibition of LINGO-1 promotes spinal cord remyelination in an experimental model of autoimmune encephalitis (17). Moreover, inhibition of LINGO-1 has been shown to enhance survival, structure, and function of dopaminergic neurons in Parkinson disease models (18). Although the function of LINGO-1 has been intensively studied, much less is known about its downstream signaling.To gain insight into the mechanisms by which LINGO-1 functions, it is of considerable importance to identify new binding partners of LINGO-1. Therefore, using the intracellular domain of LINGO-1 as bait, we employed yeast two-hybrid screening on a brain cDNA library and identified several candidates that interact with LINGO-1, one of which is the protein kinase WNK1.WNKs (with no lysine [K]) are a distinct subfamily of serine-threonine kinases, which are characterized by a unique placement of the lysine that is involved in binding ATP and catalyzing phosphoryl transfer (19). Thus far, WNKs are known composed of four members, WNK1, WNK2, WNK3, and WNK4. Mutations in the serine-threonine kinases WNK1 and WNK4 cause a Mendelian disease PAHII, featuring hypertension and hyperkalemia (20, 21), and their roles in the regulation of electrolyte flux in the kidney have been well established (22). Recently, other important features of WNKs are beginning to be understood. WNKs have also been proposed functioning in a number of non-transport processes, including cell growth, differentiation, and apoptosis (23–26). Although WNK1 has been shown to be expressed in brain (27, 28), little is known about its function in the nervous system until recently; mutations of a nervous system-specific exon of the WNK1 gene were found to cause Hereditary sensory and autonomic neuropathy type II (HSANII) (29). In this study WNK1 was demonstrated to interact with LINGO-1 and regulate Nogo-induced inhibition of neurite extension.     

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.     

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.     

Intrinsic and extrinsic inhibition of oligodendrocyte development by rat retina

Cell patterning in the vertebrate CNS reflects the combination of localized cell induction, migration and differentiation. A striking example of patterning is the myelination of visual system. In many species, retinal ganglion cell axons are myelinated in the optic nerve but are unmyelinated in the retina. Here, we confirm that rat and mouse retina lack oligodendrocytes and their precursors and identify multiple mechanisms that might contribute to their absence. Soluble cues from embryonic retina inhibit the induction of oligodendrocytes from neural stem cells and their differentiation from optic nerve precursors. This inhibition is mediated by retinal-derived BMPs. During development BMPs are expressed in the retina and addition of the BMP antagonist Noggin reversed retinal inhibition of oligodendrocyte development. The lack of retinal oligodendrocytes does not simply reflect expression of BMPs, since no oligodendrocytes or their precursors developed when embryonic retinal cells were grown in the presence of Noggin and/or inductive cues such as Shh and IGF-1. Similarly, injection of Noggin into the postnatal rat eye failed to induce oligodendrocyte differentiation. These data combined with the proposed inhibition of OPC migration by molecules selectively expressed at the nerve retina junction suggest that multiple mechanisms combine to suppress retinal myelination during development.     

CNTF is a major protective factor in demyelinating CNS disease: a neurotrophic cytokine as modulator in neuroinflammation

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). So far, immunological mechanisms responsible for demyelination have been the focus of interest. However, mechanisms regulating axon maintenance as well as glial precursor-cell proliferation and oligodendrocyte survival might also influence disease outcome. The cytokine ciliary neurotrophic factor (CNTF), which was originally identified as a survival factor for isolated neurons, promotes differentiation, maturation and survival of oligodendrocytes. To investigate the role of endogenous CNTF in inflammatory demyelinating disease, we studied myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in CNTF-deficient and wild-type C57BL/6 mice. Disease was more severe in CNTF-deficient mice and recovery was poor, with a 60% decrease in the number of proliferating oligodendrocyte precursor cells (OPCs) and a more than 50% increase in the rate of oligodendrocyte apoptosis. In addition, vacuolar dystrophy of myelin and axonal damage were more severe in CNTF-deficient mice. These specific pathological features could be prevented by treatment with an antiserum against tumor necrosis factor-alpha, suggesting that endogenous CNTF may counterbalance this effect of TNF-alpha (ref. 7). Here we identify a factor that modulates, in an inflammatory environment, glial cell survival and is an outcome determinant of EAE.     

The regional distribution of myelin oligodendrocyte glycoprotein (MOG) in the developing rat CNS: an in vivo immunohistochemical study

Using an immunohistochemical approach we have characterized the in vivo developmental distribution of myelin oligodendrocyte glycoprotein within the rat CNS. Myelin oligodendrocyte glycoprotein expression emerged in a non-uniform manner during the first 3 postnatal weeks. Although it was absent throughout the CNS of the newborn rat at postnatal day 0(P0), it had appeared in the spinal cord and brainstem by P7. The forebrain and cerebellum remained devoid of immunoreactivity until after P14. Myelin oligodendrocyte glycoprotein emerged at different times within the closely associated fasciculi of the dorsal funiculus. It appeared in the fasciculus cuneatus during the first postnatal week and in the fasciculus gracilis and corticospinal tracts during weeks 2 and 3 respectively. Myelin oligodendrocyte glycoprotein expression developed along a caudo-rostral gradient from spinal cord to forebrain and along an antero-posterior gradient within the CNS in general. The relationship between the onset of myelin oligodendrocyte glycoprotein expression and myelinogenesis was also investigated. In most regions, myelin oligodendrocyte glycoprotein expression lagged behind the initial appearance of myelin basic protein and Luxol Fast Blue-stained myelin by at least 1 week. These observations support the idea that myelin oligodendrocyte glycoprotein is the latest myelin protein to appear in development, only being expressed during the final stages of oligodendrocyte differentiation. Furthermore, the pattern of staggered expression within the dorsal columns indicates that localized, region-specific interactions may comprise a key element in the control of the terminal phases of oligodendrocyte differentiation.     

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.     

PDGF is Required for Remyelination-Promoting IgM Stimulation of Oligodendrocyte Progenitor Cell Proliferation

Background

Promotion of remyelination is a major goal in treating demyelinating diseases such as multiple sclerosis (MS). The recombinant human monoclonal IgM, rHIgM22, targets myelin and oligodendrocytes (OLs) and promotes remyelination in animal models of MS. It is unclear whether rHIgM22-mediated stimulation of lesion repair is due to promotion of oligodendrocyte progenitor cell (OPC) proliferation and survival, OPC differentiation into myelinating OLs or protection of mature OLs. It is also unknown whether astrocytes or microglia play a functional role in IgM-mediated lesion repair.

Methods

We assessed the effect of rHIgM22 on cell proliferation in mixed CNS glial and OPC cultures by tritiated-thymidine uptake and by double-label immunocytochemistry using the proliferation marker, Ki-67. Antibody-mediated signaling events, OPC differentiation and OPC survival were investigated and quantified by Western blots.

Results

rHIgM22 stimulates OPC proliferation in mixed glial cultures but not in purified OPCs. There is no proliferative response in astrocytes or microglia. rHIgM22 activates PDGFαR in OPCs in mixed glial cultures. Blocking PDGFR-kinase inhibits rHIgM22-mediated OPC proliferation in mixed glia. We confirm in isolated OPCs that rHIgM22-mediated anti-apoptotic signaling and inhibition of OPC differentiation requires PDGF and FGF-2. We observed no IgM-mediated effect in mature OLs in the absence of PDGF and FGF-2.

Conclusion

Stimulation of OPC proliferation by rHIgM22 depends on co-stimulatory astrocytic and/or microglial factors. We demonstrate that rHIgM22-mediated activation of PDGFαR is required for stimulation of OPC proliferation. We propose that rHIgM22 lowers the PDGF threshold required for OPC proliferation and protection, which can result in remyelination of CNS lesions.