Blocking LINGO-1 in vivo reduces degeneration and enhances regeneration of the optic nerve.

BACKGROUND: Two ongoing phase II clinical trials (RENEW and SYNERGY) have been developed to test the efficacy of anti-LINGO-1 antibodies in acute optic neuritis and relapsing forms of multiple sclerosis, respectively. Across a range of experimental models, LINGO-1 has been found to inhibit neuron and oligodendrocyte survival, axon regeneration, and (re)myelination. The therapeutic effects of anti-LINGO-1 antibodies on optic nerve axonal loss and regeneration have not yet been investigated. OBJECTIVE: In this series of studies we investigate if LINGO-1 antibodies can prevent acute inflammatory axonal loss, and promote axonal regeneration after injury in rodent optic nerves. METHODS: The effects of anti-LINGO-1 antibody on optic nerve axonal damage were assessed using rodent myelin oligodendrocyte glycoprotein experimental autoimmune encephalomyelitis (EAE), and its effects on axonal regeneration were assessed in optic nerve crush injury models. RESULTS: In the optic nerve, anti-LINGO-1 antibody therapy was associated with improved optic nerve parallel diffusivity measures on MRI in mice with EAE and reduced axonal loss in rat EAE. Both anti-LINGO-1 antibody therapy and the genetic deletion of LINGO-1 reduced nerve crush-induced axonal degeneration and enhanced axonal regeneration. CONCLUSION: These data demonstrate that LINGO-1 blockade is associated with axonal protection and regeneration in the injured optic nerve.

Endogenously regulated Dab2 worsens inflammatory injury in experimental autoimmune encephalomyelitis.

BACKGROUND: Neuroinflammation regulates both disease pathogenesis and repair in multiple sclerosis. In early multiple sclerosis lesion development, neuroinflammation causes demyelination and axonal injury, the likely final common determinant of disability. Here we report the identification of a novel neuroinflammatory mediator, Disabled-2 (Dab2). Dab2 is an intracellular adaptor protein with previously unknown function in the central nervous system. RESULTS: We report that Dab2 is up-regulated in lesional macrophages/microglia in the spinal cord in murine experimental autoimmune encephalomyelitis, a model of multiple sclerosis. We demonstrate that dab2 expression is positively correlated with experimental autoimmune encephalomyelitis disease severity during the acute disease phase. Furthermore, dab2-deficient mice have a less severe experimental autoimmune encephalomyelitis disease course and suffer less neuroinflammation and less axonal injury than their wild-type littermates. We demonstrate that dab2 expression is strongly associated with the expression of inducible nitric oxide synthase. We further demonstrate that Dab2 is expressed at the protein level by macrophages in early acute human multiple sclerosis lesions and that this correlates with axonal injury. CONCLUSIONS: Together, these results suggest that endogenous Dab2 exacerbates central nervous system inflammation, potentially acting to up-regulate reactive oxygen species expression in macrophages and microglia, and that it is of potential pathogenic relevance in Multiple Sclerosis.

Oligodendroglial expression of TrkB independently regulates myelination and progenitor cell proliferation.

The neurotrophin brain-derived neurotrophic factor (BDNF) has been implicated in regulating CNS myelination. BDNF mutant mice exhibit a hypomyelinating phenotype, and BDNF exerts distinct effects upon oligodendroglial proliferation, differentiation, and myelination in vitro. To investigate the precise influence that BDNF exerts in regulating CNS myelination in vivo, we have generated conditional knock-out mice in which TrkB has been deleted specifically in oligodendrocytes. Deletion of TrkB disrupted normal oligodendrocyte myelination, resulting in a significant reduction in myelin protein expression and myelination of CNS white matter tracts during development. Importantly, conditional knock-out mice exhibited normal numbers of mature oligodendrocytes and normal numbers of myelinated axons; however, myelin thickness was significantly reduced during development. These data indicate that while TrkB expression in oligodendrocytes plays no role in the initial contact with axons, it exerts an important influence in subsequent stages to promote myelin ensheathment. The conditional knock-out mice also exhibited an increased density of oligodendrocyte progenitor cells (OPCs) in CNS white matter tracts. Concordant with these results, in vitro analyses using OPCs subjected to TrkB knockdown also revealed increased OPC proliferation. Our data suggested this effect was dependent upon TrkC and p75 expression. Thus, our data demonstrate that TrkB expression in oligodendroglia exerts a direct effect on oligodendrocytes to promote myelination and an indirect effect upon the OPC population, modifying their proliferative potential.

EphA4 receptor tyrosine kinase is a modulator of onset and disease severity of experimental autoimmune encephalomyelitis (EAE).

The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS) is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE), axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections. EphA4 knockout mice exhibited a markedly less severe clinical course than wildtype mice, with a lower maximum disease grade and a slightly later onset of clinical symptoms. Numbers of infiltrating T cells and macrophages, the number and size of the lesions, and the extent of astrocytic gliosis were similar in both genotypes; however, EphA4 knockout mice appeared to have decreased axonal pathology. Blocking of EphA4 in wildtype mice by administration of soluble EphA4 (EphA4-Fc) as a decoy receptor following induction of EAE produced a delay in onset of clinical symptoms; however, most mice had clinical symptoms of similar severity by 22 days, indicating that EphA4 blocking treatment slowed early EAE disease evolution. Again there were no apparent differences in histopathology. To determine whether the role of EphA4 in modulating EAE was CNS mediated or due to an altered immune response, MOG primed T cells from wildtype and EphA4 knockout mice were passively transferred into naive recipient mice and both were shown to induce disease of equivalent severity. These results are consistent with a non-inflammatory, CNS specific, deleterious effect of EphA4 during neuroinflammation that results in axonal pathology.

Gas6 increases myelination by oligodendrocytes and its deficiency delays recovery following cuprizone-induced demyelination.

Multiple sclerosis (MS) is a complex demyelinating disease of the central nervous system. Current research has shown that at least in some cases, the primary insult in MS could be directed at the oligodendrocyte, and that the earliest immune responses are primarily via innate immune cells. We have identified a family of receptor protein tyrosine kinases, known as the TAM receptors (Tyro3, Axl and Mertk), as potentially important in regulating both the oligodendrocyte and immune responses. We have previously shown that Gas6, a ligand for the TAM receptors, can affect the severity of demyelination in mice, with a loss of signalling via Gas6 leading to decreased oligodendrocyte survival and increased microglial activation during cuprizone-induced demyelination. We hypothesised TAM receptor signalling would also influence the extent of recovery in mice following demyelination. A significant effect of the absence of Gas6 was detected upon remyelination, with a lower level of myelination after 4 weeks of recovery in comparison with wild-type mice. The delay in remyelination was accompanied by a reduction in oligodendrocyte numbers. To understand the molecular mechanisms that drive the observed effects, we also examined the effect of exogenous Gas6 in in vitro myelination assays. We found that Gas6 significantly increased myelination in a dose-dependent manner, suggesting that TAM receptor signalling could be directly involved in myelination by oligodendrocytes. The reduced rate of remyelination in the absence of Gas6 could thus result from a lack of Gas6 at a critical time during myelin production after injury. These findings establish Gas6 as an important regulator of both CNS demyelination and remyelination.

Influence of methylprednisolone on magnetic resonance and histological measures during cuprizone-induced demyelination.

MRI is widely used for routine assessment of the progression of white matter injury while patients receive therapeutic agents, such as the glucocorticoid agonist methylprednisolone (MP). Given this, it is important to determine whether MRI parameters are altered by MP treatment in the absence of changes in cellular and myelin pathology. In this study, we compared magnetic resonance and histological measures during myelin injury in mice with and without short duration MP administration. Mice were scanned with a 4.7T MRI scanner before and after MP or vehicle injections using T2WI and DTI sequences and histology was performed on the brains following the second scan. Comparison of post-injection to pre-injection MRI showed a reduced T2WI intensity in the CC and an attenuated response in ADC|| and ADC perpendicular in the MP group in comparison with the vehicle group. However, quantitative analyses of myelin staining, neurofilament intensity and oligodendrocyte and microglial density were not different between the MP and the vehicle groups, indicating that the short duration MP treatment did not alter cellular and myelin pathology. These data suggest that MP could confound the validity of paraclinical measures such as ADC|| and ADC perpendicular that are otherwise being touted as markers of either axonal integrity or myelin repair.

Modulation of bone morphogenic protein signalling alters numbers of astrocytes and oligodendroglia in the subventricular zone during cuprizone-induced demyelination.

The adult subventricular zone (SVZ) is a potential source of precursor cells to replace neural cells lost during demyelination. To better understand the molecular events that regulate neural precursor cell responsiveness in this context we undertook a microarray and quantitative PCR based analysis of genes expressed within the SVZ during cuprizone-induced demyelination. We identified an up-regulation of the genes encoding bone morphogenic protein 4 (BMP4) and its receptors. Immunohistochemistry confirmed an increase in BMP4 protein levels and also showed an increase in phosphorylated SMAD 1/5/8, a key component of BMP4 signalling, during demyelination. In vitro analysis revealed that neural precursor cells isolated from demyelinated animals, as well as those treated with BMP4, produce more astrocytes. Similarly, there were increased numbers of astrocytes in vivo within the SVZ during demyelination. Intraventricular infusion of Noggin, an endogenous antagonist of BMP4, during cuprizone-induced demyelination reduced pSMAD1/5/8, decreased astrocyte numbers and increased oligodendrocyte numbers in the SVZ. Our results suggest that lineage commitment of SVZ neural precursor cells is altered during demyelination and that BMP signalling plays a role in this process.

Gas6 deficiency increases oligodendrocyte loss and microglial activation in response to cuprizone-induced demyelination.

The TAM family of receptor protein tyrosine kinases comprises three known members, namely Tyro3, Axl, and Mer. These receptors are widely expressed in the nervous system, including by oligodendrocytes, the cell type responsible for myelinating the CNS. We examined the potential role of the TAM family and of their principle cognate ligand, Gas6 (growth arrest gene 6), in modulating the phenotype of the cuprizone model of demyelination. We found that the expression profiles of Axl, Mer, and Gas6 mRNA were increased in the corpus callosum in a temporal profile correlating with the increased migration and proliferation of microglia/macrophages in this model. In contrast, expression of Tyro3 decreased, correlating with the loss of oligodendrocytes. Gas6 both promoted in vitro survival of oligodendrocytes (39.3 +/- 3.1 vs 11.8 +/- 2.4%) and modulated markers of activation in purified cultures of microglia (tumor necrosis factor alpha mRNA expression was reduced approximately 48%). In Gas6-/- mice subjected to cuprizone-challenge, demyelination was greater than in control mice, within the rostral region of the corpus callosum, as assessed by luxol fast blue staining (myelination reduced by 36%) and by ultrastructural analysis. An increased loss of Gst-pi (glutathione S-transferase-pi)-positive oligodendrocytes was also identified throughout the corpus callosum of Gas6-/- mice. Microglial marker expression (ionized calcium-binding adapter molecule 1) was increased in Gas6-/- mice but was restricted to the rostral corpus callosum. Therefore, TAM receptor activation and regulation can independently influence both oligodendrocyte survival and the microglial response after CNS damage.

Leukemia inhibitory factor signaling modulates both central nervous system demyelination and myelin repair.

Leukemia inhibitory factor (LIF) receptor signaling limits the severity of inflammatory demyelination in experimental autoimmune encephalomyelitis, a T-cell dependent animal model of multiple sclerosis (MS) [Butzkueven et al. (2002) Nat Med 8:613-619]. To identify whether LIF exerts direct effects within the central nervous system to limit demyelination, we have studied the influence of LIF upon the phenotype of mice challenged with cuprizone, a copper chelator, which produces a toxic oligodendrocytopathy. We find that exogenously administered LIF limits cuprizone-induced demyelination. Knockout mice deficient in LIF exhibit both potentiated demyelination and oligodendrocyte loss after cuprizone challenge, an effect that is ameliorated by exogenous LIF, arguing for a direct beneficial effect of endogenous LIF receptor signaling. Numbers of oligodendrocyte progenitor cells in cuprizone-challenged mice are not influenced by either exogenous LIF or LIF deficiency, arguing for effects directed to the differentiated oligodendrocyte. Studies on the influence of LIF upon remyelination after cuprizone challenge fail to reveal any significant effect of exogenous LIF. The LIF-knockout mice do, however, display impaired remyelination, although oligodendrocyte replenishment, previously identified to occur from the progenitor pool, is not significantly compromised. Thus endogenous LIF receptor signaling is not only protective of oligodendrocytes but can also enhance remyelination, and exogenous LIF has therapeutic potential in limiting the consequences of oligodendrocyte damage.

MRI identification of the rostral-caudal pattern of pathology within the corpus callosum in the cuprizone mouse model.

PURPOSE: To characterize and compare histological and MRI-based changes within the corpus callosum (CC) in the cuprizone mouse model of multiple sclerosis (MS). MATERIALS AND METHODS: A total of 12 C57/BL6 mice were fed cuprizone from eight weeks of age for four weeks. One cohort of six cuprizone and two control mice were scanned with a T2-weighted (T2W) sequence. The other cohort of six cuprizone and four control mice were scanned using a dual-echo sequence for T2-mapping and a diffusion-weighted sequence with two orthogonal diffusion encoding directions to calculate water diffusivities parallel and perpendicular to the CC fiber (apparent diffusion coefficients [ADC](parallel) and ADC(perpendicular)). After the mice were killed, the rostral-caudal pattern of CC demyelination and other pathologies were examined using Luxol Fast Blue, neurofilament staining, and immunohistochemistry for microglia and were correlated with MRI. RESULTS: In contrast to control mice, T2W imaging (T2WI) hyperintensity, reduced ADC(parallel), and elevated ADC(perpendicular) were detected in the CC of cuprizone-fed mice, particularly in the caudal segment. The T2 value was increased in the entire CC. Marked demyelination, as well as axonal injury, microglia accumulation, and cellular infiltration were found in the caudal section of the cuprizone mouse CC. The rostral-caudal pattern of abnormalities within the CC in MRI measurements correlated well with histopathological findings. CONCLUSION: Noninvasive MRI using quantitative T2 and ADC mapping accurately characterized the rostral-caudal pattern of CC demyelination and other pathologies in cuprizone challenged mice, and thus could provide an effective way to assess the structural response to experimental therapeutics being designed for the treatment of MS.