Melissa Gresle

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 α mRNA expression was reduced ∼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-π (glutathione S-transferase-π)-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.

MR diffusion changes correlate with ultra-structurally defined axonal degeneration in murine optic nerve

Diffusion weighted imaging (DWI) and diffusion tensor imaging (DTI) are widely used to investigate central nervous system (CNS) white matter structure and pathology. Changes in principal diffusivities parallel and perpendicular to nerve fibers or axonal tracts have been associated with axonal pathology and de/dysmyelination respectively. However, the ultra-structural properties and the pathological alterations of white matter responsible for diffusivity changes have not been fully elucidated. We examined the relationship between the directional diffusivities and ultra-structural properties in mouse optic nerve using healthy animals, and mice with optic neuritis (ON) that exhibited marked inflammatory changes and moderately severe axonal pathology. Progressive axonal degeneration in ON resulted in a 23% reduction of parallel diffusivity as detected by diffusion MRI (P<10(-5)), but no change in perpendicular diffusivity. Parallel diffusion changes were highly correlated with the total axolemmal cross-sectional area in the pre-chiasmal portion of the optic nerve (r=0.86, P<0.001). This study provides quantitative evidence that reduced parallel diffusivity in the optic nerve correlates significantly with axolemmal cross-sectional area reductions. MRI-based assessment of axonal degeneration in murine ON is feasible and potentially useful for monitoring of neuro-protective therapies in preclinical trials in animals.

Neuroprotection in multiple sclerosis: A therapeutic challenge for the next decade

Multiple sclerosis (MS) is the commonest cause of progressive neurological disability amongst young, Caucasian adults. MS is considered to be an auto-immune disease that results from an attack against myelin, the layer which surrounds axons. The pathophysiology of MS is complex, with both demyelination and axonal degeneration contributing to what is essentially an inflammatory neurodegenerative disease. Axonal loss is increasingly being accepted as the histopathological correlate of neurological disability. Currently, the underpinnings of neurodegeneration in MS, and how to promote neuroprotection are only partly understood. No established treatments that directly reduce nervous system damage or enhance its repair are currently available. Moreover, the ability of currently available immunomodulatory therapies used to treat MS, such as interferon-beta, to prevent long-term disability is uncertain. Results from short-term randomized-controlled trials suggest a partial benefit with regards to disability outcomes, but this is yet to be established in long-term studies. Novel neuroprotective agents have been identified in preclinical studies but their development is being hampered by the absence of appropriate clinical platforms to test them. In this article, we will discuss some of the principal therapeutic candidates that could provide neuroprotection in MS and emerging methodologies by which to test them.

Validation of a novel biomarker for acute axonal injury in experimental autoimmune encephalomyelitis

In multiple sclerosis, inflammatory axonal injury is a key pathological mechanism responsible for the development of progressive neurological dysfunction. The injured axon represents a therapeutic target in this disease; however, therapeutic trials of neuroprotective candidates will initially require preclinical testing in an animal model of inflammatory axonal injury and subsequently the development of a reliable paraclinical measure of axonal degeneration in humans. In the present study, we demonstrate the validity of serum phosphorylated neurofilament H (pNF‐H) as a marker of axonal injury in murine experimental autoimmune encephalomyelitis (EAE). At the time of maximum disease severity (EAE day 22), the average serum pNF‐H level reached 5.7 ng/ml, correlating significantly with the EAE paraplegia score (r = 0.75, P < 0.001). On average, 40% of axons in the spinal cord were lost in EAE, and serum pNF‐H levels were highly correlated with axon loss (r = 0.8, P < 0.001). Axonal injury was a severe and acute event, insofar as serum pNF‐H levels were not significantly elevated at early (EAE day 12) or late (EAE days 35 and 50) disease time points. Our results demonstrate that acute inflammatory axonal injury is a pathological feature of murine MOG35–55 EAE, indicating that this model may mirror the acute pathological events in active multiple sclerosis lesions. Furthermore, we have validated the serum pNF‐H assay as an unbiased measurement of axonal injury in EAE, facilitating rapid screening of potential neuroprotective therapies in this model.

Injury to axons and oligodendrocytes following endothelin-1-induced middle cerebral artery occlusion in conscious rats

Injury to axons and oligodendrocytes has been poorly characterized in most animal models of stroke, and hence has been difficult to target therapeutically. It is therefore necessary to characterize axonal and oligodendroglial injury in these models, in order to rationally design putative protective compounds that minimize this injury. This study aims to characterize injury to axons and oligodendrocytes in the endothelin-1 (ET-1) model of middle cerebral artery occlusion (MCAO) in conscious rats. Transient forebrain ischemia was induced in conscious adult male Long Evans rats by the perivascular microinjection of ET-1. Quantitative histopathology was performed on forebrain sections at 6, 24, 48 and 72 h after ET-1 administration, using ballistic light analyses and immunohistochemistry for amyloid precursor protein (APP), SMI32, and Tau-1. Ballistic light analyses of cortical and striatal lesions revealed that the infarct volume was maximal in these regions by 6 h. APP and SMI32 immunohistochemistry demonstrated that axonal injury was maximal by 6 h in this model; however, some injured axons appeared to maintain good structural integrity up to 72 h after insult. Density measurements for Tau-1-immunopositive oligodendrocytes were significantly elevated within the corpus callosum from 48 h, but reductions in total oligodendrocyte numbers were not apparent up 72 h after ET-1 injection. These results indicate that axonal and oligodendroglial injury should be investigated as potential targets for delayed therapeutic intervention after MCAO.

Vaginally Administered PEGylated LIF Antagonist Blocked Embryo Implantation and Eliminated Non-Target Effects on Bone in Mice

Female-controlled contraception/HIV prevention is critical to address health issues associated with gender inequality. Therefore, a contraceptive which can be administered in tandem with a microbicide to inhibit sexually transmitted infections, is desirable. Uterine leukemia inhibitory factor (LIF) is obligatory for blastocyst implantation in mice and associated with infertility in women. We aimed to determine whether a PEGylated LIF inhibitor (PEGLA) was an effective contraceptive following vaginal delivery and to identify non-uterine targets of PEGLA in mice. Vaginally-applied 125I-PEGLA accumulated in blood more slowly (30 min vs 10 min) and showed reduced tissue and blood retention (24 h vs 96 h) compared to intraperitoneal injection in mice. Vaginally-applied PEGLA blocked implantation. PEGLA administered by intraperitoneal injection inhibited bone remodelling whereas vaginally-applied PEGLA had no effect on bone. Further, PEGLA had no effect in an animal model of multiple sclerosis, experimental auto-immune encephalomyelitis, suggesting PEGLA cannot target the central nervous system. Vaginally-administered PEGLA is a promising non-hormonal contraceptive, one which could be delivered alone, or in tandem with a microbicide. Vaginal application reduced the total dose of PEGLA required to block implantation and eliminated the systemic effect on bone, showing the vagina is a promising site of administration for larger drugs which target organs within the reproductive tract.

Neurofilament proteins as body fluid biomarkers of neurodegeneration in multiple sclerosis.

Biomarkers of axonal degeneration have the potential to improve our capacity to predict and monitor neurological outcome in multiple sclerosis (MS) patients. Neurofilament proteins, one of the major proteins expressed within neurons and axons, have been detected in cerebrospinal fluid and blood samples from MS patients and are now being actively investigated for their utility as prognostic indicators of disease progression in MS. In this paper, we summarize the current literature on neurofilament structure, assembly, and degeneration and discuss their potential utility as biomarkers for monitoring neurological decline in MS. We also discuss the need to further develop sensitive methods for assaying neurofilaments in blood to improve clinical applicability.

The MS risk allele of CD40 is associated with reduced cell-membrane bound expression in antigen presenting cells: Implications for gene function

Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS.

Leukemia Inhibitory Factor Protects Axons in Experimental Autoimmune Encephalomyelitis via an Oligodendrocyte-Independent Mechanism

Leukemia inhibitory factor (LIF) and Ciliary Neurotrophic factor (CNTF) are members of the interleukin-6 family of cytokines, defined by use of the gp130 molecule as an obligate receptor. In the murine experimental autoimmune encephalomyelitis (EAE) model, antagonism of LIF and genetic deletion of CNTF worsen disease. The potential mechanism of action of these cytokines in EAE is complex, as gp130 is expressed by all neural cells, and could involve immuno-modulation, reduction of oligodendrocyte injury, neuronal protection, or a combination of these actions. In this study we aim to investigate whether the beneficial effects of CNTF/LIF signalling in EAE are associated with axonal protection; and whether this requires signalling through oligodendrocytes. We induced MOG35–55 EAE in CNTF, LIF and double knockout mice. On a CNTF null background, LIF knockout was associated with increased EAE severity (EAE grade 2.1±0.14 vs 2.6±0.19; P<0.05). These mice also showed increased axonal damage relative to LIF heterozygous mice, as indicated by decreased optic nerve parallel diffusivity on MRI (1540±207 µm2−/s vs 1310±175 µm2−/s; P<0.05), and optic nerve (−12.5%) and spinal cord (−16%) axon densities; and increased serum neurofilament-H levels (2.5 fold increase). No differences in inflammatory cell numbers or peripheral auto-immune T-cell priming were evident. Oligodendrocyte-targeted gp130 knockout mice showed that disruption of CNTF/LIF signalling in these cells has no effect on acute EAE severity. These studies demonstrate that endogenous CNTF and LIF act centrally to protect axons from acute inflammatory destruction via an oligodendrocyte-independent mechanism.

Serum phosphorylated neurofilament-heavy chain levels in multiple sclerosis patients

Objectives We evaluated whether the measurement of serum phosphorylated neurofilament heavy chain (pNF-H) titre is likely to be a valid biomarker of axonal injury in multiple sclerosis (MS). Methods Serum pNF-H concentrations were measured by ELISA in cases with relapsing-remitting (RR)-MS (n=81), secondary progressive (SP) MS (n=13) and primary progressive (PP)-MS; n=6) MS; first demyelinating event (FDE; n=82); and unaffected controls (n=135). A subset of MS cases (n=45) were re-sampled on one or multiple occasions. The Multiple Sclerosis Severity Score (MSSS) and MRI measures were used to evaluate associations between serum pNF-H status, disease severity and cerebral lesion load and activity. Results We confirmed the presence of pNF-H peptides in serum by ELISA. We showed that a high serum pNF-H titre was detectable in 9% of RR-MS and FDE cases, and 38.5% of SP-MS cases. Patients with a high serum pNF-H titre had higher average MSSS scores and T2 lesion volumes than patients with a low serum pNF-H titre. Repeated sampling of a subset of MS cases showed that pNF-H levels can fluctuate over time, likely reflecting temporal dynamics of axonal injury in MS. Conclusions A subset of FDE/MS cases was found to have a high serum pNF-H titre, and this was associated with changes in clinical outcome measures. We propose that routine measurement of serum pNF-H should be further investigated for monitoring axonal injury in MS.