A step towards earlier detection of multiple sclerosis

Related publications: Shaharabani R., Ram-On M., Talmon Y. & Beck R. Pathological transitions in myelin membranes driven by environmental and multiple sclerosis conditions. Proc. Natl. Acad. Sci. 115, 11156–11161 (2018). DOI: 10.1073/pnas.1804275115; Shaharabani R., Ram-On M., Avinery R., Aharoni R., Arnon R., Talmon Y. & Beck R. Structural Transition in Myelin Membrane as Initiator of Multiple Sclerosis. J. Am. Chem. Soc. 138, 12159–12165 (2016). DOI: 10.1021/jacs.6b04826

Publication keywords: SAXS; Phase transition; Myelin; Multiple sclerosis; Lamellar phase; Inverted hexagonal phase; Membrane; Lipids 

Using Diamond Light Source’s Small Angle Scattering & Diffraction beamline (I22), I911-SAXS at MAX IV Laboratory, SWING at SOLEIL synchrotron, and P12 at EMBL BioSAXS, a team of researchers from Tel Aviv University and the Technion-Israel Institute of Technology mapped, for the first time, the delicate and complicated force balance between the myelin sheath constituents and their effect on the myelin structure.

It is well known that fatty, membranous materials (such as lipids) can organise into a variety of shapes (phases), including stacked sheets (lamellae), tubes, or cubes, to name a few. Controlling these phases is essential to proper function. This new information will allow the researchers to identify critical components involved in neurodegenerative diseases such as multiple sclerosis (MS). 

MS is an autoimmune disease resulting in the destruction of myelin, a fatty substance that insulates nerves and increases the speed at which signals travel between nerve cells. MS affects more than 2.3 million people worldwide and has no cure. By investigating the microscopic structure of myelin membrane under various conditions, the researchers were able to identify the critical conditions that alter the myelin structure. Their results showed a phase transition from a healthy stack of lamellas to a diseased inverted hexagonal phase as a result of the altered lipid stoichiometry, myelin basic protein content, and environmental conditions such as salinity and temperature. Myelin lipid composition, and the physiological environmental conditions, are critical for myelin to function properly, and the results demonstrate that these conditions should be contemplated as alternative routes for MS early detection. 

 
Figure 1: Schematic summary of the conditions resulting in phase transition in healthy and
diseased myelin.
Figure 1: Schematic summary of the conditions resulting in phase transition in healthy and diseased myelin.

The myelin sheath, a repeating lipid-protein multilamellar structure, acts as an electrical insulator by forming a capacitor surrounding the axon, insuring fast nerve conduction1. The myelin membrane is composed of multiple lipid types, and the myelin basic protein (MBP). MBP facilitates binding of the myelin sheath to the underlying cytoplasmic membrane presumably through a charge-charge interaction where the positively-charged protein binds to the negatively-charged membranes acting as an intermolecular adhesion glue2,3.

Multiple sclerosis (MS) is an autoimmune disease resulting in myelin destruction. Currently, over 2.3 million people are affected by MS, while its etiology or cure are still elusive2,3. Previous in vivo studies correlated MS with changes in lipid composition and MBP deficiency3. Using Small Angle X-ray Scattering (SAXS), we recently evaluated the structural consequences of lipid and protein compositions at altered environmental conditions4,5. In particular, the structures of healthy (i.e. normal lipid composition) and diseased myelin (i.e. modified lipid composition) states were addressed (Fig. 1).
 
In Fig. 2 we show high-resolution synchrotron SAXS patterns, taken at I22, of normal and modified lipid compositions with and without the inclusion of MBP. The scattering profiles are composed of Bragg reflections overlaid on the membrane form-factor signal. Multilamellar and hexagonal phases reflections are found at wave-vectors:
and respectively. Here, n, h, and k are the Miller integers, dL and aH represent the real-space unit-cell length of the lamellar and hexagonal phase respectively.
 
In the absence of MBP, normal lipid composition (Fig. 2, red line) results in one lamellar phase with unit-cell spacing of dL = 113 Å, while the modified lipid composition (Fig. 2, black line) results in coexistence of lamellar and inverted hexagonal (HII) phases with unit-cell lengths of dL = 123 and aH = 90 Å respectively4. The addition of MBP, however, results in phase transition for both lipid compositions. In the modified lipid composition (Fig. 2, blue line), the HII phase quickly demolished and the SAXS signal results in a single lamellar phase with a unit-cell length of dL = 84 Å (above 7 wt% MBP content). For the normal lipid composition (Fig. 2, green line), the one lamellar phase in the absence of the protein transitions into three correlation peaks, indicating three unit-cell distances.
 
The dominant peak corresponds to the unit-cell length of the membrane (above 7 wt% MBP, dL = 88 Å, green arrow in Fig. 2), while the two shallow peaks correspond to in-plane MBP organisation within the membrane leaflets (Fig. 2, orange and pink arrows). These findings highlight that MBP is crucial for stabilising the membrane to form functional lamellar structures. Moreover, MBP causes the leaflets to adhere to one another, and flattens the membranes, counteracting local negative curvatures. In the absence of the protein, we expect that the normal lipid composition will be susceptive to structural phase transitions due to local changes in the membrane curvature.
 
Figure 2: Normal and modified lipid compositions with and without MBP. Normal lipid
composition only (red curve), normal lipid composition with 20% w/w MBP (green curve),
modified lipid composition only (black curve), modified lipid composition with 20% w/w
MBP (blue curve). For clarity of representation the scattering patterns are shifted in the
intensity-axis.
Figure 2: Normal and modified lipid compositions with and without MBP. Normal lipid composition only (red curve), normal lipid composition with 20% w/w MBP (green curve), modified lipid composition only (black curve), modified lipid composition with 20% w/w MBP (blue curve). For clarity of representation the scattering patterns are shifted in the intensity-axis.

In vivo, the environmental conditions are highly regulated and controlled. Potassium and sodium are essential for proper action potential along the myelinated axons. Calcium and zinc ions have the same oxidation state, yet they have different biological functions in the body, such as signal transduction pathways, and the induction of protein-protein interactions. Due to the fact that the myelin membrane is close to a structural phase transition, we addressed the effects of altered environmental conditions on the cytoplasmic myelin membrane structures5

Changing the monovalent ion type or its concentration results in structural changes above a critical concentration, C* (Fig. 3a-b). Above C*, the lamellar unit-cell length (dL) decreases with increasing concentration for both monovalent ions (Fig. 3a-b, square symbols). In contrast, the hexagonal phase unit-cell length (aH) shows an opposite trend (Fig. 3a-b, hexagonal symbols), where aH increases slightly with increasing monovalent salt concentration (Fig. 3a). This phenomenon occurs for both monovalent salts, and persists until C*, at which point aH becomes salt independent, and the lamellar phase is well ordered. Importantly, exchanging sodium with potassium ions exhibits the same pathological coexisting HII phase even for healthy lipid composition above (Fig. 3b).
 
Figure 3: Normal and modified lipid compositions for different divalent ions concentration
(C). Modified (a, c) and normal (b, d) lipid concentration for different monovalent (a, b) and
divalent (c, d) ion concentrations. Dashed lines indicate C*.
Figure 3: Normal and modified lipid compositions for different divalent ions concentration (C). Modified (a, c) and normal (b, d) lipid concentration for different monovalent (a, b) and divalent (c, d) ion concentrations. Dashed lines indicate C*.

The divalent ions results are dramatically different. Here, we find that the structures are much more ion-specific (Fig. 3c-d). For example, the inverted hexagonal phases differ in the unit-cell length sizes at saturation with aH (Mg2+) > aH (Ca2+) > aH (Zn2+). Moreover, above a critical concentration, C*(Zn2+) = 5 mM < C*(Ca2+) = 7 mM < C*(Mg2+) = 9 mM, we find a coexisting dense lamellar phase (dd). This new phase has the same unit-cell length for all ion types and lipid compositions (dd ∼ 64 Å). Moreover, above C*, we find no changes in the hexagonal unit-cell lengths (Fig. 3, hexagonal symbols). Surprisingly, here we find that even the normal lipid composition exhibits coexisting of inverted hexagonal phases, similar to the diseased state.

In order to study myelin vulnerability, we measured its structure in the context of membrane self-assembly by SAXS. From a physical perspective, the competing forces between the lipids determine the nanoscopic structure and the macroscopic mesophase, discussed in detail in ref4,5. In these studies, we identified several factors that tend to destabilise the lamellar phase, and induce the formation of the inverted hexagonal phase. Minor alterations of the environmental conditions can drive structural instabilities and the formation of the HII phase. Lower salinity and low temperature are favorable for healthy lamellar phase up to about physiological condition.

In summary, even healthy myelin lipid composition is on the verge of a structural phase transition. The phase transition can be linked as a possible trigger for the outbreak of MS. In the process of phase transition from lamellar into the HII phase, the membrane undergoes large undulations typical to the increase in local curvatures5. These undulations can induce spontaneous pores, resulting in the vulnerability of the membrane to an attack by the immune system. Therefore, changes in lipid composition, depletion of MBP, or local environmental modification near the myelin, whether by ion type or ion concentration, can result in pathological phase transition that characterises the diseased state.
 

References:

  1. Wood D. D. et al. Is the myelin membrane abnormal in multiple sclerosis? J. Membr. Biol. 79, 195–201 (1984). DOI: 10.1007/BF01871058
  2. Ohler B. et al. Role of lipid interactions in autoimmune demyelination. Biochim. Biophys. Acta - Mol. Basis Dis. 1688, 10–17 (2004). DOI: 10.1016/j.bbadis.2003.10.001
  3. Boggs J. M. et al. Interaction forces and adhesion of supported myelin lipid bilayers modulated by myelin basic protein. Proc. Natl. Acad. Sci. 106, 3154–3159 (2009). DOI: 10.1073/pnas.0813110106
  4. Shaharabani R. et al. Structural Transition in Myelin Membrane as Initiator of Multiple Sclerosis. J. Am. Chem. Soc. 138, 12159–12165 (2016). DOI: 10.1021/jacs.6b04826
  5. Shaharabani R. et al. Pathological transitions in myelin membranes driven by environmental and multiple sclerosis conditions. Proc. Natl. Acad. Sci. 115, 11156–11161 (2018). DOI: 10.1073/pnas.1804275115
Funding acknowledgement:
The work was supported by the Israel Science Foundation (571/11 and 550/15), and the Sackler Institute for Biophysics and the Abramson Center for Medical Physics at Tel Aviv University. Travel grants to the synchrotron were provided by BioStruct-X.
 
Corresponding authors:
Rona Shaharabani, Tel Aviv University, ronas@tauex.tau.ac.il and Roy Beck, Tel Aviv University, roy@tauex.tau.ac.il