Vanderbilt Institute of Chemical Biology



Discovery at the VICB







New Insights Into Myelin Formation



By: Carol A. Rouzer, VICB Communications
Published:  July 19, 2017


Structural studies of peripheral myelin protein 22 reconstituted in lipid vesicles reveal a natural tendency to form myelin-like structures.


Rapid transmission of nerve impulses in both the central nervous system (CNS) and peripheral nervous system (PNS) relies on the presence of myelin, a complex membranous structure that surrounds neuronal axons and serves as insulation. Myelin is formed from extensions of the membranes of glial cells (oligodendrocytes in the CNS or Schwann cells in the PNS), which wrap round the axon in a spiral fashion (Figure 1). As the spiral forms, the layers of the membrane compact into a tight coating of protein and lipid (Figure 2). Each section of myelin is separated from the next by a node where the flow of ions across unmyelinated axonal plasma membrane transmits the nerve impulse. It is this jumping of neuronal transmission from node to node that is responsible for a marked increase in the speed of the signal. Myelin-forming membrane exhibits a distinct lipid composition rich in cholesterol and sphingolipids, and it is notable for high concentrations of specific proteins. In the PNS, these include myelin protein zero (MPZ), peripheral myelin protein 22 (PMP22), and myelin basic protein (MBP). This unique composition must be responsible for the myelin's ability to both wrap around an axon and form compact layers. However, the mechanism by which this occurs is not fully understood. Now, Vanderbilt Institute of Chemical Biology member Chuck Sanders, his collaborators Melanie Ohi (Dept. of Cell & Developmental Biology) and Jun Li (Dept. of Neurology), and their laboratories report on the ability of PMP22 to form myelin-like structures in lipid vesicles in vitro [K. F. Mittendorf, J. T. Marinko, et al., (2017), Sci. Adv., 3, e1700220].


FIGURE 1. Diagrammatic representation of the process of myelination. This example is given for myelination in the CNS by oligodendrocytes; however it is similar to myelination in the PNS by Schwann cells. The cell wraps layers of membrane in a spiral around the axon, and then the lipids are compacted. Channels between the compacted areas enable a flow of cytoplasmic constituents to reach the end of the spiral. Figure reproduced by permission from Macmillan Publishers Ltd. from K.-J. Changi, et al., (2016), Nat. Neurosci., 19, 190. Copyright 2016.



FIGURE 2. Diagrammatic representation of myelin compaction in the CNS. The process is similar in the PNS. Compaction proceeds from the outer to the inner layers. A key component in both the CNS and PNS is myelin basic protein (MBP), which closely binds two apposed membranes, extruding uncompacted myelin components [such as cyclic nucleotide phosphodiesterase (CNP) shown here]. Figure reproduced by permission from Macmillan Publishers Ltd. from K.-J. Changi, et al., (2016), Nat. Neurosci., 19, 190. Copyright 2016.



PMP22 is an integral membrane protein that traverses the membrane four times (Figure 3). Accounting for 2 - 5% of PNS myelin proteins by mass, PMP22 is required for proper myelination of peripheral nerves. Trisomy of the gene for PMP22 leads to Charcot-Marie-Tooth disease Type 1A (CMT1A), while heterozygous deletion of the gene produces hereditary neuropathy with liability to pressure (HNPP). CMT1E results from dominant heterozygous missense mutations in the gene. Although varying in severity, all of these conditions are degenerative neuropathies characterized by abnormal myelin in the PNS. Thus, it is evident that the presence of the correct amount of structurally sound PMP22 is necessary for PNS myelination.



FIGURE 3. Outline of the structure of PMP22, showing the four transmembrane helices and the two extracellular loops (ECL1 and ECL2). Key highly conserved residues are highlighted in blue, and cysteine residues are outlined in red. Figure reproduced under the Creative Commons Attribution-NonCommercial 4.0 International License from K. F. Mittendorf, J. T. Marinko, et al., (2017), Sci. Adv., 3, e1700220.


In initial studies, the investigators attempted to obtain two-dimensional crystals of PMP22 that had been reconstituted in phospholipid/sphingomyelin vesicles. They did not obtain usable crystals, but examination of the preparation by negative stain electron microscopy revealed something unexpected. The membranous vesicles had formed multilayered assemblies that strongly resembled myelin, particularly the myelin formed in calanoid copepods – planktonic crustaceans in which the myelin sheath is formed from the neuronal cell membrane rather than glial cells. The structures, which they named myelin-like assemblies (MLAs), appeared to be compressed vesicles that were stacked and wrapped around each other in a horseshoe-like shape when examined more carefully by cryo-electron microscopy and cryo-tomography (Figures 4 & 5). They were distinct from the concentric nested membranes characteristic of multilamellar vesicles (MLVs) that were also present in the preparation, along with distorted MLAs, clumped vesicles, and aggregates of proteins and/or lipids. These findings indicated that PMP22 can induce flattening and wrapping of membranes into horseshoe-shaped stacks. The investigators noted that these stacks are particularly similar to those observed during myelin formation in copepods.



FIGURE 4. Cryo-electron micrograph showing two MLVs (A & B) and two MLAs (C & D). MLVs comprise concentric nested vesicles, while MLAs are tightly apposed layers of flattened vesicles wrapped into a horseshoe shape.  Figure reproduced under the Creative Commons Attribution-NonCommercial 4.0 International License from K. F. Mittendorf, J. T. Marinko, et al., (2017), Sci. Adv., 3, e1700220.




FIGURE 5. Line drawings of the structures of MLAs (A) and MLVs (B). Figure reproduced under the Creative Commons Attribution-NonCommercial 4.0 International License from K. F. Mittendorf, J. T. Marinko, et al., (2017), Sci. Adv., 3, e1700220.



Reconstitution of PMP22 in lipid vesicles at varying protein:lipid ratios clearly demonstrated that MLA formation requires the correct ratio of the two components. This is reminiscent of human inherited neuropathies that result from expression of too much or too little PMP22. The investigators hypothesized that PMP22 can undergo trans-homophilic interaction – a protein-protein interaction between two PMP22 molecules on different membranes – that would cause the vesicles to adhere to one another. They initially thought that cysteine disulfide bond formation might be involved in such an interaction, but vesicles reconstituted with PMP22 in which all four cysteine residues had been mutated to alanine were able to form MLAs as well as those reconstituted with the wild-type protein.


Prior research had suggested that the two extracellular loops of PMP22 (ECL1 and ECL2, Figure 3) could be the sites of a trans-homophilic interaction. Support for this hypothesis came from experiments in which MLA formation was conducted in the presence of glutathione S-transferase (GST) fusion ECL1 or ECL2 oligopeptides. In both cases, the peptides inhibited MLA formation. The ECL2-derived oligopeptide was the more potent of the two, exhibiting a maximum inhibition of 98% as compared to 60% for the ECL1-derived peptide. Further studies demonstrated that selected mutations of key amino acids in these loops could also lead to a loss of ability of PMP22 to promote MLA formation when reconstituted into vesicles. Also of interest was the L16P mutation located in the first transmembrane domain. This mutation is associated with the Trembler-J disease phenotype, with severe neuropathy in humans. L16P PMP22 produced loosely packed, disorganized MLAs when reconstituted into lipid vesicles.


The investigators note that PMP22 is probably incorporated bidirectionally into their lipid vesicles. In other words, the ECLs face outward from both sides of the membrane. This differs from the situation found in cells where all PMP22 is oriented so that the ECLs project into the extracellular space. Thus, in this in vitro model, trans-homophilic interactions could cause adhesion of both sides of each membrane bilayer, while in cells, they could only promote adhesion between the extracellular leaflets. Interesting support for a potential role of PMP22 in membrane adhesion comes from studies in HeLa cells where expression of the recombinant protein leads to bridging between the cells.


These findings provide important insight into a potential role for PMP22 in myelin formation and/or structural integrity. They suggest that PMP22 alone assists in establishing membrane curvature and adhesion characteristic of the myelin sheath. Obviously, other proteins also play a role, but these studies lay an important foundation for exploration of those roles. In fact, it would be interesting to see if other myelin-associated proteins exhibit distinctive properties in this in vitro system.




View Sciences Advances article: Peripheral myelin protein 22 alters membrane architecture










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