LINGO-1 Regulates Oligodendrocyte Differentiation through the Cytoplasmic Gelsolin Signaling Pathway.

Differentiation and maturation of oligodendrocyte progenitor cells (OPCs) involve the assembly and disassembly of actin microfilaments. However, how actin dynamics are regulated during this process remains poorly understood. Leucine-rich repeat and Ig-like domain-containing Nogo receptor interacting protein 1 (LINGO-1) is a negative regulator of OPC differentiation. We discovered that anti-LINGO-1 antibody-promoted OPC differentiation was accompanied by upregulation of cytoplasmic gelsolin (cGSN), an abundant actin-severing protein involved in the depolymerization of actin filaments. Treating rat OPCs with cGSN siRNA reduced OPC differentiation, whereas overexpression of cGSN promoted OPC differentiation in vitro and remyelination in vivo Furthermore, coexpression of cGSN and LINGO-1 blocked the inhibitory effect of LINGO-1. Our study demonstrates that cGSN works downstream of LINGO-1 signaling pathway, which enhances actin dynamics and is essential for OPC morphogenesis and differentiation. This finding may lead to novel therapeutic approaches for the treatment of demyelinating diseases such as multiple sclerosis (MS).SIGNIFICANCE STATEMENT Myelin loss and subsequent axon degeneration contributes to a variety of neurological diseases, such as multiple sclerosis (MS). Understanding the regulation of myelination by oligodendrocytes is therefore critical for developing therapies for the treatment of MS. We previously demonstrated that leucine-rich repeat and Ig-like domain-containing Nogo receptor interacting protein 1 (LINGO-1) is a negative regulator of oligodendrocyte differentiation and that anti-LINGO-1 promotes remyelination in preclinical animal models for MS and in a phase II acute optic neuritis clinical trial (RENEW). The mechanism by which LINGO-1 regulates oligodendrocyte differentiation is unknown. Here, we demonstrate that LINGO-1 regulates oligodendrocyte differentiation and maturation through the cytoplasmic gelsolin signaling pathway, providing new drug targets for the treatment of demyelination diseases.

Identification and control of a poliomyelitis outbreak in Xinjiang, China.

BACKGROUND: The last case of infection with wild-type poliovirus indigenous to China was reported in 1994, and China was certified as a poliomyelitis-free region in 2000. In 2011, an outbreak of infection with imported wild-type poliovirus occurred in the province of Xinjiang. METHODS: We conducted an investigation to guide the response to the outbreak, performed sequence analysis of the poliovirus type 1 capsid protein VP1 to determine the source, and carried out serologic and coverage surveys to assess the risk of viral propagation. Surveillance for acute flaccid paralysis was intensified to enhance case ascertainment. RESULTS: Between July 3 and October 9, 2011, investigators identified 21 cases of infection with wild-type poliovirus and 23 clinically compatible cases in southern Xinjiang. Wild-type poliovirus type 1 was isolated from 14 of 673 contacts of patients with acute flaccid paralysis (2.1%) and from 13 of 491 healthy persons who were not in contact with affected persons (2.6%). Sequence analysis implicated an imported wild-type poliovirus that originated in Pakistan as the cause of the outbreak. A public health emergency was declared in Xinjiang after the outbreak was confirmed. Surveillance for acute flaccid paralysis was enhanced, with daily reporting from all public and private hospitals. Five rounds of vaccination with live, attenuated oral poliovirus vaccine (OPV) were conducted among children and adults, and 43 million doses of OPV were administered. Trivalent OPV was used in three rounds, and monovalent OPV type 1 was used in two rounds. The outbreak was stopped 1.5 months after laboratory confirmation of the index case. CONCLUSIONS: The 2011 outbreak in China showed that poliomyelitis-free countries remain at risk for outbreaks while the poliovirus circulates anywhere in the world. Global eradication of poliomyelitis will benefit all countries, even those that are currently free of poliomyelitis.

An outbreak following importation of wild poliovirus in Xinjiang Uyghur Autonomous Region, China, 2011.

BACKGROUND: After more than 10 years without a case of wild poliovirus (WPV) in China, an outbreak occurred in 2011 in Xinjiang Uyghur Autonomous Region. METHODS: Acute flaccid paralysis (AFP) case surveillance was strengthened with epidemiological investigations and specimen collection and serological surveys were conducted among hospitalized patients. RESULTS: There were 21 WPV cases and 23 clinical compatible polio cases reported. WPV was isolated from 14 contacts of AFP cases and 13 in the healthy population. Incidence of WPV and clinical compatible polio cases were both highest among children <1 years, however, 24/44 (54.5%) polio cases were reported among adults aged 15-39 years. CONCLUSIONS: High coverage of routine immunization should be maintained among children until WPV transmission is globally eradicated. Expansion of AFP case surveillance and use of serologic surveys to estimate population immunity should be conducted rapidly to guide preparedness and response planning for future WPV outbreaks.

LINGO-1 regulates oligodendrocyte differentiation by inhibiting ErbB2 translocation and activation in lipid rafts.

Oligodendrocyte differentiation is negatively regulated by LINGO-1 and positively regulated by the ErbB2 receptor tyrosine kinase. In wild-type oligodendrocytes, inhibition of ErbB2 blocks differentiation, whereas activation of ErbB2 promotes differentiation. In LINGO-1(-/-) oligodendrocytes, inhibition of ErbB2 blocks oligodendrocyte differentiation; whereas activation of ErbB2 does not enhance differentiation. Biological and biochemical evidence showing that LINGO-1 can directly bind to ErbB2, block ErbB2 translocation into lipid rafts, and inhibit its phosphorylation for activation. The study demonstrates a novel regulatory mechanism of ErbB2 function whereby LINGO-1 suppresses oligodendrocyte differentiation by inhibiting ErbB2 translocation and activation in lipid rafts.

Structure of the LINGO-1-anti-LINGO-1 Li81 antibody complex provides insights into the biology of LINGO-1 and the mechanism of action of the antibody therapy.

Multiple sclerosis (MS) is an autoimmune-inflammatory disease of the central nervous system (CNS) with prominent demyelination and axonal injury. While most MS therapies target the immunologic response, there is a large unmet need for treatments that can promote CNS repair. LINGO-1 (leucine-rich repeat and Ig-containing Nogo receptor interacting protein-1) is a membrane protein selectively expressed in the CNS that suppresses myelination, preventing the repair of damaged axons. We are investigating LINGO-1 antagonist antibodies that lead to remyelination as a new paradigm for treatment of individuals with MS. The anti-LINGO-1 Li81 antibody,BIIB033, is currently in clinical trials and is the first MS treatment targeting CNS repair. Here, to elucidate the mechanism of action of the antibody, we solved the crystal structure of the LINGO-1-Li81 Fab complex and used biochemical and functional studies to investigate structure-function relationships. Li81 binds to the convex surface of the leucine-rich repeat domain of LINGO-1 within repeats 4-8. Fab binding blocks contact points used in the oligomerization of LINGO-1 and produces a stable complex containing two copies each of LINGO-1 and Fab that results from a rearrangement of contacts stabilizing the quaternary structure of LINGO-1. The formation of the LINGO-1-Li81 Fab complex masks functional epitopes within the Ig domain of LINGO-1 that are important for its biologic activity in oligodendrocyte differentiation. These studies provide new insights into the structure and biology of LINGO-1 and how Li81 monoclonal antibody can block its function.

Enhanced surveillance of acute flaccid paralysis following importation of wild poliovirus in Xinjiang Uygur Autonomous Region, China.

BACKGROUND: After being polio free for more than 10 years, an outbreak occurred in China in 2011 in Xinjiang Uygur Autonomous Region (Xinjiang) following the importation of wild poliovirus (WPV) originating from neighboring Pakistan. METHODS: To strengthen acute flaccid paralysis (AFP) surveillance in Xinjiang, "zero case daily reporting" and retrospective searching of AFP cases were initiated after the confirmation of the WPV outbreak. To pinpoint all the polio cases in time, AFP surveillance system was expanded to include persons of all ages in the entire population in Xinjiang. RESULTS: Totally, 578 AFP cases were reported in 2011 in Xinjiang, including 21 WPV cases, 23 clinical compatible polio cases and 534 non-polio AFP cases. Of the 44 polio cases, 27 (61.4%) cases were reported among adults aged 15-53 years. Strengthening AFP surveillance resulted in an increase in the number of non-polio AFP cases in 2011 (148 children < 15 years) compared with 76 cases < 15 years in 2010. The AFP surveillance system in Xinjiang was sensitive enough to detect polio cases, with the AFP incidence of 3.28/100,000 among children < 15 years of age. CONCLUSIONS: Incorporating adult cases into the AFP surveillance system is of potential value to understand the overall characteristics of the epidemic and to guide emergency responses, especially in countries facing WPV outbreak following long-term polio free status. The AFP surveillance system in Xinjiang was satisfactory despite limitations in biological sample collection.

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.

American literature news narration based on computer web technology.

Driven by internet technology, online has become the main way of news dissemination, but redundant information such as navigation bars and advertisements affects people's access to news content. The research aims to enable users to obtain pure news content from redundant web information. Firstly, based on the narrative characteristics of literary news, the Term Frequency-Inverse Document Frequency (TF-IDF) algorithm is employed to extract pure news content from the analyzed web pages. The algorithm uses keyword matching, text analysis, and semantic processing to determine news content's boundaries and key information. Secondly, the news text classification algorithm (support vector machine, K-nearest neighbor, AdaBoost algorithm) is selected through comparative experiments. The news extraction system based on keyword feature and extended Document Object Model (DOM) tree is constructed. DOM technology analyzes web page structure and extracts key elements and information. Finally, the research can get their narrative characteristics by studying the narrative sequence and structure of 15 American literary news reports. The results reveal that the most used narrative sequence in American literary news is sequence and flashback. The narrative duration is dominated by the victory rate and outline, supplemented by scenes and pauses. In addition, 53.3% of the narrative structures used in literary news are time-connected. This narrative structure can help reporters have a clear conceptual structure when writing, help readers quickly grasp and understand the context of the event and the life course of the protagonists in the report, and increase the report's readability. This research on the narrative characteristics of American literature news can provide media practitioners with a reference on news narrative techniques and strategies.

Role of transmembrane semaphorin Sema6A in oligodendrocyte differentiation and myelination.

Myelination is regulated by extracellular proteins, which control interactions between oligodendrocytes and axons. Semaphorins are repulsive axon guidance molecules, which control the migration of oligodendrocyte precursors during normal development and possibly in demyelinating diseases. We show here that the transmembrane semaphorin 6A (Sema6A) is highly expressed by myelinating oligodendrocytes in the postnatal mouse brain. In adult mice, Sema6A expression is upregulated in demyelinating lesions in cuprizone-treated mice. The analysis of the optic nerve and anterior commissure of Sema6A-deficient mice revealed a marked delay of oligodendrocyte differentiation. Accordingly, the development of the nodes of Ranvier is also transiently delayed. We also observed an arrest in the in vitro differentiation of purified oligodendrocytes lacking Sema6A, with a reduction of the expression level of Myelin Basic Protein. Their morphology is also abnormal, with less complex and ramified processes than wild-type oligodendrocytes. In myelinating co-cultures of dorsal root ganglion neurons and purified oligodendrocytes we found that myelination is perturbed in absence of Sema6A. These results suggest that Sema6A might have a role in myelination by controlling oligodendrocyte differentiation.

Sphingosine 1-phosphate receptor modulator fingolimod (FTY720) does not promote remyelination in vivo.

The sphingosine 1-phosphate (S1P) receptor modulators have emerged as a new therapeutic opportunity paradigm for the treatment of immune-mediated demyelinating diseases such as multiple sclerosis (MS). The S1P analog fingolimod (FTY720) has been shown to alleviate disease burden in immune-mediated animal models of MS, and has been approved for treatment in clinical trials in patients with MS in the United States. While the immunological effects of FTY720 are well established, there is controversy in the literature regarding the contribution of FTY720 on myelin repair. Here, we directly assessed the impact of FTY720 on myelin repair in cuprizone and lysolecithin (LPC) demyelination models that have a minimal immunological component. FTY720 failed to promote remyelination in either animal model. These studies suggest that while FTY720 may be effective at modulating the immunological attack in MS, it may benefit from an add-on therapy to enhance the myelin repair required for long-term functional restoration in MS.

Oligodendrocyte differentiation and myelination defects in OMgp null mice.

OMgp is selectively expressed in CNS by oligodendrocyte. However, its potential role(s) in oligodendrocyte development and myelination remain unclear. We show that OMgp null mice are hypomyelinated in their spinal cords, resulting in slower ascending and descending conduction velocities compared to wild-type mice. Consistent with the hypomyelination, in the MOG induced EAE model, OMgp null mice show a more severe EAE clinical disease and slower nerve conduction velocity compared to WT animals. The contribution of OMgp to oligodendrocyte differentiation and myelination was verified using cultured oligodendrocytes from null mice. Oligodendrocytes isolated from OMgp null mice show a significant decrease in the number of MBP(+) cells and in myelination compared to wild-type mice. The dramatic effects of the OMgp KO in oligodendrocyte maturation in vivo and in vitro reveal a new and important function for OMgp in regulating CNS myelination.

Comparison of the Design Space of Products with Different Initial Saturation.

Vacuum freeze-drying is a promising technology widely used in pharmaceuticals. Preparing products with prebuilt porosity has attracted considerable attention due to its potential in shortening process duration. However, the design space for the primary drying of initially unsaturated products remains unclear. A novel index, average power, was proposed in this paper to represents the collapse risk. And a multiphase model was employed in this paper to build the design space for the products with initial voids. The simulation results show that both the drying time and average power show higher sensitivity to the temperature variation than pressure. In addition, the initial saturation has significant impacts on the design space, with small initial saturation resulting in vast design space and vice versa, which implies that small initial saturation is more beneficial for the actual production. This paper would be helpful for the development of freeze-drying.

Exposure levels of anti-LINGO-1 Li81 antibody in the central nervous system and dose-efficacy relationships in rat spinal cord remyelination models after systemic administration.

LINGO-1 (leucine-rich repeat and Ig domain containing NOGO receptor interacting protein-1) is a negative regulator of myelination and repair of damaged axons in the central nervous system (CNS). Blocking LINGO-1 function leads to robust remyelination. The anti-LINGO-1 Li81 antibody is currently being evaluated in clinical trials for multiple sclerosis (MS) and is the first MS therapy that directly targets myelin repair. LINGO-1 is selectively expressed in brain and spinal cord but not in peripheral tissues. Perhaps the greatest concern for Li81 therapy is the limited access of the drug to the CNS. Here, we measured Li81 concentrations in brain, spinal cord, and cerebral spinal fluid in rats after systemic administration and correlated them with dose-efficacy responses in rat lysolecithin and experimental autoimmune encephalomyelitis spinal cord models of remyelination. Remyelination was dose-dependent, and levels of Li81 in spinal cord that promoted myelination correlated well with affinity measurements for the binding of Li81 to LINGO-1. Observed Li81 concentrations in the CNS of 0.1 to 0.4% of blood levels are consistent with values reported for other antibodies. To understand the features of the antibody that affect CNS penetration, we also evaluated the pharmacokinetics of Li81 Fab2, Fab, and poly(ethylene glycol)-modified Fab. The reagents all showed similar CNS exposure despite large differences in their sizes, serum half-lives, and volumes of distribution, and area under the curve (AUC) measurements in the CNS directly correlated with AUC measurements in serum. These studies demonstrate that exposure levels achieved by passive diffusion of the Li81 monoclonal antibody into the CNS are sufficient and lead to robust remyelination.

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.

Troy/Taj and its role in CNS axon regeneration.

Binding of myelin inhibitors to the NgR1/p75/LINGO-1 signaling complex activates RhoA to mediate the inhibition of axonal outgrowth. The nerve growth factor receptor p75, a TNF family receptor, is absent or poorly expressed in certain types of neurons that respond to myelin inhibitors, thereby prompting speculation that other TNF family receptors are involved in the NgR1 complex. Troy/Taj is an orphan TNF family receptor that is broadly expressed in postnatal and adult neurons. Troy binds to NgR1 and can functionally replace p75 in the p75/NgR1/LINGO-1 complex to activate RhoA and block neurite outgrowth in the presence of myelin inhibitors. Neurons from Troy-deficient mice are more resistant to the suppressive action of the myelin inhibitors. The discovery of TROY function in axon growth is an important step for understanding the complex regulation of axonal regeneration by diverse members of the TNF receptor family.

Dissecting demyelination.

The loss of central nervous system myelin and the failure of remyelination by oligodendrocytes contribute to the functional impairment that characterizes diseases such as multiple sclerosis. Why myelin repair fails in multiple sclerosis is currently unclear; however, new understanding of the generation of oligodendrocytes and myelination during development, as well as an increasing understanding of the bases of successful remyelination, are providing new insights and therapeutic targets. We propose that successful myelin repair of the adult CNS recapitulates a sequence of stages that generally correlate with those seen during development, whereas unsuccessful myelin repair results from the perturbation of a critical process in any one of several sequential events. Defining the rate-limiting steps and most vulnerable aspects at each stage of myelin repair will provide logical targets for therapeutic intervention in demyelinating diseases such as multiple sclerosis.

Discovery of Potent Selective Nonzinc Binding Autotaxin Inhibitor BIO-32546.

Autotaxin (ATX) is a lysophospholipase D that is the main enzyme responsible for generating LPA in body fluids. Although ATX was isolated from a conditioned medium of melanoma cells, later it was discovered to play a critical role in vascular and neuronal development. ATX has also been implicated in primary brain tumor, fibrosis, and rheumatoid arthritis, as well as neurological diseases such as multiple sclerosis, Alzheimer's disease, and neuropathic pain. As ATX and LPA levels are increased upon neuronal injury, a selective ATX inhibitor could provide a new approach to treat neuropathic pain. Herein we describe the discovery of a novel series of nonzinc binding reversible ATX inhibitors, particularly a potent, selective, orally bioavailable, brain-penetrable tool compound BIO-32546, as well as its synthesis, X-ray cocrystal structure, pharmacokinetics, and in vivo efficacy.

LINGO-1 negatively regulates TrkB phosphorylation after ocular hypertension.

The antagonism of LINGO-1, a CNS-specific negative regulator of neuronal survival, was shown to promote short-term survival of retinal ganglion cell (RGC) in an ocular hypertension model. LINGO-1 antagonists, combined with brain-derived neurotrophic factor (BDNF), can increase the length of neuron survival through an unclear molecular mechanism. To determine the relationship between LINGO-1 and BDNF/TrkB receptor in neuronal protection, we show here that LINGO-1 forms a receptor complex with TrkB and negatively regulates its activation in the retina after ocular hypertension injury. LINGO-1 antagonist antibody 1A7 or soluble LINGO-1 (LINGO-1-Fc) treatment upregulates phospho-TrkB phosphorylation and leads to RGC survival after high intraocular pressure injury. This neuronal protective effect was blocked by anti-BDNF antibody. LINGO-1 antagonism therefore promotes RGC survival by regulating the BDNF and TrkB signaling pathway after ocular hypertension.

Promotion of central nervous system remyelination by induced differentiation of oligodendrocyte precursor cells.

OBJECTIVE: Repair of demyelinated axons in diseases such as multiple sclerosis requires activation of the myelination program in existing or newly recruited oligodendrocyte precursor cells (OPCs). The control of OPC differentiation and initiation of myelination during repair is poorly understood. In this study, we test the ability of anti-LINGO-1 reagents to promote myelination in vitro and remyelination in the rodent adult central nervous system in vivo. METHODS: The effects of LINGO-1 antagonists on the differentiation of OPCs and the promotion of myelination has been assayed using a combination of coculture and slice culture preparations. Using three different animal models of demyelination and remyelination, we morphologically and functionally assessed the effects of LINGO-1 antagonists on OPC differentiation and myelin repair. RESULTS: The data indicate that in vitro treatment with antagonists of LINGO-1 promote OPC differentiation and myelination, whereas in vivo remyelination is accelerated in lysophosphatidylcholine- or cuprizone-induced demyelination. This remyelination is associated with enhanced OPC differentiation and functional recovery of conduction velocities in demyelinated axons. INTERPRETATION: Our studies demonstrate that LINGO-1 antagonism promotes OPC differentiation and remyelination, and suggest LINGO-1 functions as an inhibitor of OPC differentiation to retard central nervous system remyelination.

Characterization of trisulfide modification in antibodies.

Trisulfides are a posttranslational modification formed by the insertion of a sulfur atom into a disulfide bond. Although reports for trisulfides in proteins are limited, we find that they are a common modification in natural and recombinant antibodies of all immunoglobulin G (IgG) subtypes. Trisulfides were detected only in interchain linkages and were predominantly in the light-heavy linkages. Factors that lead to trisulfide formation and elimination and their impact on activity and stability were investigated. The peptide mapping methods developed for characterization and quantification of trisulfides should be applicable to any antibody and can be easily adapted for other types of proteins.