Innovations in Treatments for Multiple Sclerosis

¶ … 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 paper come from animals sacrificed at 30 days after immunization with MOG. (a) What degree of behavioural/clincal impairment would be observed in wild-type animals 7 days post-immunization? Explain your answer.

(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). According to Mi et al.

(2007: 1231), anti-LINGO-1 therapy improves axonal integrity and myelination in two ways as follows: Aside from the lower EAE scores observed in these mice and rats, physiological improvements in axonal integrity were revealed by DTI, and at the cellular level, enhanced and new myelination were revealed by histological staining and electron microscopy, respectively." 9. Anti-LINGO-1 antibodies administered systemically are able to cross the normally impermeable blood-brain barrier. Apply your understanding of the lectures to describe two different ways this barrier could be activated to become permeable to antibodies in this experimental scenario.

(2 marks) Both diffusion and osmosis could active the normally impermeable blood brain barrier to antibodies in the Mi et al. (2007) experiment. 10. The pathology of multiple sclerosis is not limited to degradation of oligodendrocytes. What are two other neuronal hallmarks of MS described in your lectures? For each of these pathologies give an explanation for its emergence. (2marks) The myelinating cells of the central nervous system are known as oligodendrocytes (Week 11A lecture notes).

At present, researchers believed that progressive serotonergic neuronal atrophy with synaptic inactivation rather than axonal degeneration is the hallmark of MS (Sandyk, 1998). This is an advance over the conceptualisations of the neurological manifestations of MS in the past that were regarded as having resulted from demyelination of axons with relative preservation of axonal integrity (Sandyk, 1998). Research has demonstrated that that axonal degeneration is also present in MS cases (Sandyk, 1998).

ASSESSMENT 3B Fundamental Neuroscience • Questions refer to the paper Ballas N, Lioy DT, Grunseich C & Mandel G (2009) Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology. Nat Neurosci;12(3):311-7 ASSESSMENT 3B QUESTIONS: 1.

Why are MeCP2 mutations always sporadic rather than hereditary? (2 marks) A member of the methyl-CpG -- binding protein family that functions as transcriptional repressors, methyl-CpG -- binding protein 2 (MeCP2) is comprised of three functional domains that preclude its transmission hereditarily: (a) the methyl DNA -- binding domain (MBD) that binds to methylated CpG dinucleotides, (b) a transcriptional repressor domain (TRD) that can recruit co-repressors and chromatin remodelling complexes, and (c) the C-terminal domain that facilitates binding to DNA (Ballas, Lioy, Grenseich & Mandel, 2009: 311).

The National Institute of Neurological Disorders and Stroke (Rett syndrome, 2015: 2) reports that, "Although Rett syndrome is a genetic disorder, less than 1% of recorded cases are inherited or passed from one generation to the next. Most cases are spontaneous, which means the mutation occurs randomly." 2. Contrast the genetic aetiology of Rett syndrome as compared to schizophrenia. Explain your answer in general terms addressing the relative involvement of genes and environment in each case (2 marks).

The research to date indicates that almost all cases of Rett syndrome are the result of a mutation in MeCP2 (Rett syndrome, 2015). According to the National Institute of Neurological Disorders and Stroke (Rett syndrome, 2015: 2), "Because the MeCP2 gene does not function properly in individuals with Rett syndrome, insufficient amounts or structurally abnormal forms of the protein are produced and can cause other genes to be abnormally expressed." By contrast, although the precise aetiology of schizophrenia remains unclear, it is known that it includes various environmental factors.

For instance, Zoler (2005: 33) notes that, "A synthesis of findings is that schizophrenia [and] schizoaffective disorder result from defects in common genes. Environmental factors that cause effects such as developmental impairment or susceptibility to social adversity help determine which specific disorder results from the underlying genetic liability." 3.

(a) Why would Rett syndrome manifest more severely in males than in females? (1 mark) In those cases where males carry a similar MeCP2 mutation to those found in females with Rett syndrome on their single X chromosome in which there are no other "normal" X chromosome to compensate, males will typically be affected more severely than females (Clark & Jaworska, 2011).

According to Clark and Jaworska (2011: 3), "It is also possible for males to manifest problems from 'milder' genetic alterations in the MECP2 gene that do not usually cause serious problems (like Rett syndrome) in females.

These less severe genetic alterations can still cause problems with development, learning and behaviour in males, however, as males do not have the stabilising effects of a normal MeCP2 gene present in each cell in addition to the mutated copy." (b) Explain which figure in this paper best models the dosage of MeCP2 expression in female patients with Rett syndrome? (2 marks) Figure 5 provides the best model of the altered morphology of wild-type neurons cultured with ACM from Mecp2 -- / y astrocytes.

This figure shows altered morphology at the single cell level. 4. "There are several RTT mouse models, each of which contains different mutations in MeCP2." Describe two ways in which a gene sequence can be disrupted and explain the consequences of this for protein translation. Do not investigate specific mutations reported Rett syndrome mutations but talk in general about changes to genetic architecture.

(2 marks) According to Gonzalez and Adams (2012), although the precise mechanisms by which MeCP2 regulates gene expression remain unclear, the research to date indicates that MeCP2 functions as a transcriptional activator or silencer and executes these activities through both genome-wide and gene-specific mechanisms. In addition, Gonzalez and Adams (2012: 2894) note that, "MeCP2 may be controlling gene expression by regulating chromatin structure and long-range chromatin interactions in addition to affecting expression through binding to proximal promoter regions." 5. Supplementary Figure S1 shows a quantitative measurement of MeCP2 expression using Western Blot analysis.

In order from highest to lowest which cell types contain the highest levels of MeCP2 protein? List all four. (1 mark) The respective levels of MeCPS protein depicted in Supplementary Figure S1 in descending order are as follows: (a) hippocampal neurons; (b) cortical neurons; (c) oligodendrocytes; and (d) oligodendrocyte progenitor cells (Ballas et al., 2009). 6. Figure 1 shows that MeCP2 is present in the nuclei of mature glia. Is this subcellular localisation consistent with the supposed role of MeCP2 discussed in the introduction? Explain why or why not.

(2 marks) The subcellular localisation of MeCP2 in the nuclei of mature glia is consistent with the introductory observations made by Ballas et al. (2009). For instance, Ballas and his associates (2009: 311) report that, "MeCP2 is present in the normal brain not only in neurons, but in all types of glia, including astrocytes, oligodendrocyte progenitor cells (OPCs) and ligodendrocytes." 7. Histones are large globular proteins that wrap the DNA double helix into bundles that are hard to access.