¶ … Neuroscience • Questions refer to the paper Mi S, Hu B, Hahm K, Luo Y et al., (2007) LINGO-1 antagonist promotes spinal cord remyelination and axonal integrity in MOG-induced experimental autoimmune encephalomyelitis. Nat Med;13(10):1228-33.

MOG-induced murine experimental autoimmune encephalomyelitis (EAE) is an experimentally-induced disease. In what ways does it model the 'outside-in' theory of multiple sclerosis? Your answer must name the correct antigens and cell types involved in the process (3 marks).

Experimental autoimmune encephalomyelitis (EAE) involves both neurological and immune components (Mi et al., 2007). EAE is formed from the CD4+ T cell -- mediated immune response targeted at specific proteins within the central nervous system (CNS) which provides a model of multiple sclerosis (MS) (Week 10 Lecture Notes). The research to date indicates that loss of the Nogo receptor -- interacting protein LINGO-1 moderates EAE by altering the generation and the infiltration into the CNS of encephalogenic T cells that are implicated in EAE pathology (Mi et al., 2007). The research to date also confirms that clinical manifestations of MOG-induced EAE mirror multiple sclerosis and are caused by a neurological component as well as an autoimmune inflammatory component that involves axon loss and demyelination (Mi et al., 2007). These researchers showed that decreased levels of functioning in LINGO-1 in rats and mice and rats caused by LINGO-1 knockout or treatment with LINGO-1 antagonist antibody is an efficacious treatment for arresting disease progression and alleviating the symptoms of clinical neuropathology that are associated with MOG-induced EAE (Mi et al., 2007).

The experiment by Mi et al. (2007) indicated that the impact of LINGO-1 antagonism is specific to the individual central nervous system because deficiencies in LINGO-1 deficiency were found to not impact the induction phase of EAE demonstrated by the release of T-cell cytokines after MOG immunization and through T-cell proliferation assays. In addition, these researchers also determined that it is possible to induce EAE through the adoptive transfer of encephalogenic T cells from MOG-immunized Lingo1-knockout mice to wild-type (WT) mice (Mi et al., 2007). Moreover, this experiment indicates that when encephalogenic T cells from MOG-immunized WT mice are transferred to Lingo1-knockout mice, EAE is mitigated, findings that are congruent with other recent studies in this area (Mi et al., 2007). Taken together, the findings that emerged from the Mi et al. (2007) study together with the other research to date lends support to the use of LINGO-1 antagonists as a novel therapeutic approach for the treatment of demyelinating diseases (Mi et al., 2007).

2. Briefly describe three specific pieces of evidence from the paper that address whether the immune/inflammatory pathway is impaired in LINGO-

1 knockout mice. (3 marks)

On page 1228, Mi et al. report that, "Blocking LINGO-1 function, either through Lingo1 knockout or through treatment with anti-LINGO-1, promotes functional recovery in the EAE model." In addition, on page 1129, Mi et al. report that, "Both wild-type (WT) and Lingo1-knockout mice developed EAE symptoms; however, EAE scores were significantly lower in Lingo1-knockout mice throughout all stages of disease progression." Likewise, on page 1129, Mi et al. note that, "Quantitative analysis of myelinated axons indicated that there were more myelinated fibers in Lingo1-knockout EAE mice than in the WT EAE controls." The aggregated data from this study indicate that LINGO-1 deficiency is most likely attributable to changed in the CNS compartment wherein remyelination is facilitated and the deficiency was shown to not affect the ability of inflammatory immune effector cells and encephalogenic T cells to cause EAE.

3. Describe one finding from this paper and one discussed in your lectures that support the 'inside-out' theory of aetiology. (3 marks)

Based on their use of an electron microscopy to demonstrate that lower EAE scores correspond to remyelination, Mi et al. (2007) also found that myelin damage was observed is the characteristic 'dying-back' oligodendrogliopathy presenting as an 'inside-out' type more frequently in the demyelinated areas of WT mice compared to those identified in knockout EAE mice.

4. How would the 'inside-out' theory explain the behaviour of the myelincontaining macrophage (blue stain) marked by arrows in the figure attached below? (2 marks)

In contrast to diseases that attack tissues and result in breakdown from the outside in, the nerves in multiple sclerosis have been shown to break down from the inside-out. The myelin-containing macrophages marked by arrows in the figure with the blue stain, the pale areas indicate demyelination is occurring and that lesions are developing from the inside to the myelin and an inside-out destructive process is taking place within these cells.

5. The neuropathological microimages throughout this...

(1 mark)
Although the study is silent concerning what degree of behavioural or clinical impairment, behavioural and clinical impairments in wild-type animals subjected to this type of experiment 7 days post-immunization would be expected to include demyelination which was assessed by Mi et al. (2007) as a percentage of the total demyelination area as measured over the total spinal cord area by the use of random sections from the fourth lumbar segment of each animal. There were damaged myelin sheaths identified in wild-type animals that were characterised by frequent separated and loose layers or degraded sheath structures; in some cases, myelin sheaths were totally absent. In addition, the analysis of the aggregated data indicates that LINGO-1 deficiency most likely causes an altered CNS compartment in which remyelination is encouraged.

(b) What degree of tissue pathology would you expect to see animals sacrificed at 7 days? (1 mark) Explain your answer with reference to both the paper and an understanding of acute multiple sclerosis.

Although the study is also silent concerning the degree of tissue pathology experienced in animals sacrificed at 7 days, it is reasonable to suggest that these animals would have diminished LINGO-1 functions. In addition, the lower EAE scores identified in the experimental animals would evince physiological improvements in axonal integrity at 7 days. At the cellular level, these animals would also likely experience decreased motor dysfunction but enhanced and new myelination. Because the effects of LINGO-1 antagonism appear to be central nervous system-specific, it would be unlikely to find changes in T-cell proliferation and the release of T-cell cytokines following MOG immunization.

6. (a) Why are the results shown in Figure 2e more dramatic than those shown in Figure 2f? (2 marks)

Figures 2e and 2f from the Mi et al. (2007) study are depicted below. Figure 2e shows that rats that were treated by local delivery of antibody before the clinical onset of EAE symptoms indicate that the fractional anisotropy values from the dorsal spinal cord areas. Figure 2f shows the dorsal spinal cord areas of spinal cords from rats treated by systemic delivery of antibody after the clinical onset of EAE symptoms and the fractional anisotropy values. The results shown in Figure 2e are more dramatic because the anti-LINGO-1 -- treated group were found to be significantly higher than those of the IgG control -- treated group.

(b) Yet why is the second of those experiments described as being more 'clinically relevant?' (1 marks)

The decreased EAE severity following anti-LINGO-1 antagonist treatment reflects improved axonal function and integrity. The LINGO-1 functional antagonist was therefore considered to have potential therapeutic efficacy irrespective of whether it is delivered locally or systemically and was found to be efficacious before or after the manifestation of EAE symptoms.

7. Mi et al., (2007) "have provided an in vivo proof-of-concept for the development of LINGO- 1 antagonists as a novel therapeutic approach for the treatment of demyelinating diseases."

(a) How are all current treatments for MS different from this one in terms of their targets and the timeframe of applicability? (2 marks)

There is no cure for multiple sclerosis and the current treatments that are available only treat the symptoms of the disorder or to slow its progression (Bishop & Tschopp, 2000). In contrast to the approach proposed by Mi et al. (2007) wherein demyelination appears to be reversed, all other current treatments for MS treat the symptoms using pharmacological or physical rehabilitation modalities (Treatment and drugs, 2015).

(b) Name an example of one of these treatments covered in the lectures and briefly describe how it works (1 mark)

One pharmacological intervention, dimethyl fumarate, was recently approved by the Food and Drug Administration (Week 10 Lecture Notes). For instance, according to Chamberlin (2013: 46), "Dimethyl fumarate oral delayed-release capsules, formerly called BG-12, were approved by FDA March 27, 2013 for the treatment of relapsing forms of multiple sclerosis (MS). It is the third oral drug to be recently approved for the treatment of these types of MS." The research to date indicates that dimethyl fumarate and its metabolite monomethyl fumarate activate an antioxidant response pathway, nuclear factor [erythroid-derived 2]-like 2 (Nrf2), in vitro and in vivo in both humans and animals (Chamberlin, 2013). It is currently believed that the Nrf2 pathway is implicated in cellular defence against oxidative stress (Chamberlin, 2013).

8. Describe two ways in which anti-LINGO-1 therapy improves axonal integrity and myelination? (2 marks).

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