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Project
The search for toxin-derived modulators of voltage-gated sodium channels and their in vivo effects in autoimmune encephalomyelitis
Voltage-gated sodium channels (Navs) are key players in the generation and propagation of electrical signals in the human body. They are found in the membrane of electro-excitable cells and underlie the event of action potentials. Due to their crucial role in excitability, malfunctioning of these channels inevitably leads to severe physiological disorders. Therefore, defect Navs are associated with diseases like epilepsy and the long QT syndrome. Recently it has been established that Navs also could play a role in the pathogenesis of multiple sclerosis (MS). This disease is hallmarked by degeneration of the myelin sheath surrounding axons,a strong inflammatory reaction and axonal loss in the CNS. Several Nav isoforms have been demonstrated to be involved in the pathology of MS. For instance, elevated levels of Nav1.2 and Nav1.6 were observed in active MS lesions. It was suggested that the increase in sodium current that this brings along, is responsible for the neuronal damage that is noticed in later stages of the disease. Block of these channels possibly could lead to neuroprotection. Owing to these results, clinical trials were set up to validate the effects of Nav blockers. However, the tested Nav blockers target a large range of Navs without any specificity. In this thesis, novel compounds were sought that specifically target the Nav isoforms mentioned to be important in MS. This was achieved by looking into the venoms of sea anemones, scorpions, solitary wasps and cone snails as it is known to contain toxins that act on specific Nav isoforms.
In the first chapter, two resembling toxins isolated from thevenom of two solitary wasps were investigated. The β-pompilidotoxin (β-PMTX) established a specific effect on Nav1.6 and was characterized further. The toxin causes a decrease of the fast activation of Nav1.6 in a dose-dependent matter. In this way, the influx of sodium ions is diminished. In addition, the toxin generates a steady-state current. This is a typical effect of toxins binding to a specific site on Navs, called site 3. However, this toxin has the unique property of combining its typical site 3-effect with the mentioned decrease of fast activation.
The venom of cone snails is also known to comprise toxins that block Navs. These toxins are called µ-conotoxins. In the second chapter, synthetic peptides were designed, based on structures of naturally µ-occurring toxins from venoms of the cone snails Conus kinoshitai and Conus bullatus. The main goal was to develop a miniaturized peptide that maintained the typical features of the original peptides. These features include i) a strong block on Navs with activities in the nanomolar range, ii) an effect on specific Nav isoforms and iii) a short sequence. The initial designed peptide, called Mini peptide, demonstrated a block on Nav1.2, Nav1.4 and Nav1.6 at micromolar concentrations. Based on this Mini peptide, a series of analogues was subsequently developed. Three peptides (Midi, R1-Midi and R2-Midi) were provento be potent blockers of Nav1.2, Nav1.4 and Nav1.6, acting at nanomolar concentrations. In addition, an NMR structure was determined forthe Midi peptide, which revealed no α-helix as a secondary structure. This was the first µ-conotoxin derivative for which this unique feature was witnessed.
In the last chapter, the in vivo effects of β-PMTX and the Midi peptide were investigated in a commonly used mouse model of MS, called experimental autoimmune encephalomyelitis. Based on the available results for non-specific Nav blockers, an amelioration of the clinical scores was expected for mice treated with the Midi peptide. Unexpectedly, the Midi peptide caused a worsening of the disease pattern in these mice. Several techniques were used to find out which cellular compounds were underlying the observed deterioration. It was demonstrated that the expression levels of Nav1.2 and Nav1.6 were elevated in brain tissues of mice treated with the Midi peptide. Although the precise origin of the deleterious effects that we saw in the mice receiving the Midi peptide remains unanswered, the results of this chapter confirm the complex roles of Navs in EAE and possibly MS. Our study could not strengthen the rationale for the use of Nav blockers for the treatment of MS. Nonetheless, it underlines that there is an urgent need to further clarify the role of Navs in the pathology of MS.
In the first chapter, two resembling toxins isolated from thevenom of two solitary wasps were investigated. The β-pompilidotoxin (β-PMTX) established a specific effect on Nav1.6 and was characterized further. The toxin causes a decrease of the fast activation of Nav1.6 in a dose-dependent matter. In this way, the influx of sodium ions is diminished. In addition, the toxin generates a steady-state current. This is a typical effect of toxins binding to a specific site on Navs, called site 3. However, this toxin has the unique property of combining its typical site 3-effect with the mentioned decrease of fast activation.
The venom of cone snails is also known to comprise toxins that block Navs. These toxins are called µ-conotoxins. In the second chapter, synthetic peptides were designed, based on structures of naturally µ-occurring toxins from venoms of the cone snails Conus kinoshitai and Conus bullatus. The main goal was to develop a miniaturized peptide that maintained the typical features of the original peptides. These features include i) a strong block on Navs with activities in the nanomolar range, ii) an effect on specific Nav isoforms and iii) a short sequence. The initial designed peptide, called Mini peptide, demonstrated a block on Nav1.2, Nav1.4 and Nav1.6 at micromolar concentrations. Based on this Mini peptide, a series of analogues was subsequently developed. Three peptides (Midi, R1-Midi and R2-Midi) were provento be potent blockers of Nav1.2, Nav1.4 and Nav1.6, acting at nanomolar concentrations. In addition, an NMR structure was determined forthe Midi peptide, which revealed no α-helix as a secondary structure. This was the first µ-conotoxin derivative for which this unique feature was witnessed.
In the last chapter, the in vivo effects of β-PMTX and the Midi peptide were investigated in a commonly used mouse model of MS, called experimental autoimmune encephalomyelitis. Based on the available results for non-specific Nav blockers, an amelioration of the clinical scores was expected for mice treated with the Midi peptide. Unexpectedly, the Midi peptide caused a worsening of the disease pattern in these mice. Several techniques were used to find out which cellular compounds were underlying the observed deterioration. It was demonstrated that the expression levels of Nav1.2 and Nav1.6 were elevated in brain tissues of mice treated with the Midi peptide. Although the precise origin of the deleterious effects that we saw in the mice receiving the Midi peptide remains unanswered, the results of this chapter confirm the complex roles of Navs in EAE and possibly MS. Our study could not strengthen the rationale for the use of Nav blockers for the treatment of MS. Nonetheless, it underlines that there is an urgent need to further clarify the role of Navs in the pathology of MS.
Date:1 Nov 2008 → 28 Sep 2012
Keywords:Multiple sclerosis
Disciplines:Laboratory medicine, Palliative care and end-of-life care, Regenerative medicine, Other basic sciences, Other health sciences, Nursing, Other paramedical sciences, Other translational sciences, Other medical and health sciences
Project type:PhD project