Topic 02

The Biochemistry of Myelin

 Myelin
 Multi-layered spiral of oligodendrocyte membrane that surrounds
axons
 Each myelin segment originates from a single oligodendrocyte

 Each oligodendrocyte contributes many myelin segments, but each

segment is apparently associated with axons from different neurons
 Chemical integration among neurons

 Glial cells can pass molecules to neurons, then a single

oligodendrocyte could pass molecules to each of the dozens of
different neurons it myelinates
 The integration of the functions of these associated neurons
 Characteristics of Myelination
 Myelination occurs predominantly just prior to and/or following birth,
depending on the species.
 In humans, myelination occurs predominantly after birth, with the
majority taking place during the first two years of life.
 the more mature an organism is at the time of birth, the greater the
extent of myelination EXAMPLES
 Arcuate fibers, which connect different portions of the cerebral cortex
to each other, are the last to be myelinated
 The sequence of myelination corresponds roughly to phylogenesis, or
the evolutionary sequence.
 PNS → Spinal Cord → Cerebral Cortex → Rest of the brain →
Arcuate fibers (can take up to 3rd decade of life)
 Benefits of Myelin
 The myelin investment around an axon is functionally
beneficial.
 myelination increases, the neuron is capable of increasing
its rate of firing.
 increasing the rate of excitation of neurons in a developing
brain increases the rate of myelination.
 The two processes, therefore, drive each other and are
interdependent.

 Myelinated axons need only have action potentials

at the nodes of Ranvier, (1 mm apart)
 Benefits of Myelin
 The myelin sheath serves to optimize the function of the

neuron in at least three ways

 First, myelin serves as an electrical insulator. In

demyelinating diseases, the loss of this insulation results

in an electrical "cross talk" between adjacent axons,

→pathologically disruptive to brain function.

 Second, myelin increases conduction velocity.

 Finally, myelin decreases the metabolic demand

necessary for the generation of an action potential.

 Benefits of Myelin
 The velocity of a conducted action potential in a myelinated
axon is directly proportional to the diameter of the axon plus the
myelin sheath.

 Unmyelinated axons (which are still surrounded by glial cell

bodies, but not by multi-layered myelin sheaths) have been
observed to conduct action potentials indirect proportion to the
square root of the axon's diameter.

 From a metabolic point of view, myelinated fibers have a three-

hundred to four-hundredfold advantage over non-myelinated
fibers with respect to their energy needs.
 Molecular Architecture of Myelin
 Myelin sheath is composed of multiple layers of
compacted plasma membranes
 It contains the same molecules as the membranes of
other cell types + myelin molecules.
unique functions in the formation of myelin layer
potential contribution to disease processes
Significant turn over rate of some myelin molecules
Some molecules function as auto antigens

 Can play a role in the development of CNS diseases.

 Cholesterol
 The most abundant molecule in myelin

 CNS cholesterol is predominantly unesterified.

 Most cholesterol in other animal membranes has a fatty acid
attached to its hydroxyl group by an ester linkage.
 This linkage is similar to that by which fatty acids are attached in
phospholipids, triacylglycerols, etc.

 In certain pathological states esterified cholesterol is found in

significant quantities in the CNS
 In Multiple sclerosis (demyelinated lessions) the percentage
of esterified cholesterol is significantly increased
 Phospholipids
 The predominant phospholipid in myelin is
phosphatidyl ethanolamine (PE)

 Phosphatidyl choline (PC) is the major

phospholipid in other tissues.

 Most phospholipids, as indeed nearly all lipids,

contain fatty acid residues

 Brain phospholipids especially PE contain alkyl

chains with different level of oxidation (fatty
aldehydes or alcohols)

 Less common in other tissues

 The PE of brain contains a large percentage Double bond occurs between the first
and second carbon, the chain behaves
plasmalogens as an aldehyde
 Phospholipids
 In the structure of phosphoglycerides. In most lipids, X is acyl, that is, R–(CO)
 In alkyl ethers, present mainly in brain ethanolamine phosphoglycerides (2–3%),
X is a long-chain hydrocarbon (C16, C18)
 For plasmalogens, which constitute about 60% of adult human brain PtdEtn, X is
1-alk-1′enyl (i.e. –CH=CH–R)

 Note that myo-inositol is written in the

d-configuration, where the 1′ position
is linked to the PtdOH moiety
 For polyphosphoinositides, additional
phosphate groups are present in the
3, 4 or 5 positions.
 Sphingolipids
 Brain tissue contains a large amount of Sphingolipids
 White-matter myelin and gray matter

 Sphingolipids,
 Contain a residue of sphingosine, (a long-chain fatty base)

 Also contain a fatty acid attached in amide linkage to the second carbon of the sphingosine

 This molecule of sphingosine plus a fatty acid is called a ceramide.

 It does not itself appear in significant quantities in nerve tissue, but it is an obligatory metabolic

intermediate for all sphingolipids.

Ceramide
 Sphingolipids
 The myelin sphingolipids are simpler in structure than those
of neurons.
 Most have only one or two residues attached to the
ceramide.
 In myelin the first carbon of the sphingosine chain is most
commonly linked to a residue of galactose.
 Myelin sphingolipids are also phospholipids,
(sphingomyelin)
 Sphingomyelin actually occurs in all mammalian
membranes
 Sphingolipids
 The three-dimensional structure of sphingomyelin
gives it considerable resemblance to phosphatidyl
choline (PC)
 These two lipids can be hydrolyzed by some of the
same enzymes
 Sphingomyelin and PC are both localized to the
outer leaflets of plasma membranes
 Sphingolipids
 Cerebroside (another most characteristic lipid of myelin )
 The first carbon of the ceramide is galactose

 Large amounts of cerebroside are present in myelin

 Synthesized after the blood-brain barrier has formed

 Relationship to the Immune system and Immune reaction

 Sulfatide, contains a sulfate residue on the 3' hydroxyl group of the

galactose of the cerebroside

 Ganglioside GM4,cerebroside has a sialic acid residue (N-acetyl

neuraminic acid, or NANA) attached to the same 3' position

 These molecule occurs characteristically in myelin

 Have a negative charge, from the sulfate or Sialic acid group

 Sphingolipids
 Sphingomyelin is not peculiar to brain and
recognized as self by the immune system.
 Negatively charged molecules are mainly
considered as self.
 Cerebroside positively charged might serve serve
as autoantigen
 Myelin or crude white matter is auto antigenic to the
same species even for identical twins
 Proteins
 White matter specific proteins of myelin can function
as autoantigens.
 The two major such molecules are myelin basic protein
(MBP) and proteolipid protein (PLP).
 MBP is not an integral membrane protein
 Often considered as the major auto-antigen of white
matter
 Found in the soluble cytoplasmic compartment of the
oligodendrocyte
 Proteolipid protein
 The proteolipid protein (PLP) is peculiar to myelin

 Predominantly synthesized after the blood-brain barrier has sequestered the

CNS from immune surveillance

 PLP has auto antigenic properties

 PLP is a highly unusual protein & quite different from MBP

 unusual structural feature is that fatty acids are attached to the protein, as esters

 this protein have many lipid-like properties and fewer protein-like properties

 PLP is soluble in organic solvents, such as chloroform and not soluble in water

 PLP is an integral membrane protein, spanning the membrane in which it

occurs

 Share some structural features in common with neurotransmitter receptors,

though no receptor function

 Immunogenicity of MBP
 MBP alone, when injected into another organism of the same species, generates an

immune response.

 MBP can be used experimentally to generate an autoimmune disease called

experimental autoimmune encephalomyelitis (EAE).

 EAE is considered as an animal model for multiple sclerosis (MS).

 EAE, when produced solely by the use of MBP, differs from multiple sclerosis in

many respects, principally in that EAE does not wax and wane as MS does

 Considerable effort to create animal models with more of the clinical features of MS.

Several recent models use a mixture of autoantigenic molecules. Such autoimmune

"cocktails" usually include MBP, cerebroside, PLP, and often a ganglioside.

 Although such cocktails can produce a CNS white-matter disease with many

features of MS,
 Ultra structure of Myelin
• Myelin, and many of its morphological
features, such as nodes of Ranvier and
Schmidt–Lantermann clefts, can be seen
readily in the light microscope
• EM Studies
– Myelin can be seen as a series of
alternating dark and less dark lines
(protein layers) separated by unstained
zones (the lipid hydrocarbon chains)
– The outer plasma membranes of the
myelin layer are not actually fused (less
Rabbit peripheral nerve (anterior root), stained
dark) with toluidine blue
– The dark, or major line is the fused, inner
protein layers of the cell membrane.
 Nodes of Ranvier
Two adjacent segments of myelin
on one axon are separated by a
node of Ranvier

At the paranodal region and the

Schmidt–Lantermann clefts the
cytoplasmic surfaces of myelin are
not compacted and Schwann or
glial cell cytoplasm is included
within the
sheath.

Schmidt–Lantermann clefts are structures

where the cytoplasmic surfaces of the myelin
sheath have not compacted to form the major
dense line and therefore contain Schwann or
glial cell cytoplasm
 Myelin is an extension of a glial plasma membrane
• Myelination in the PNS is preceded by invasion of
the nerve bundle by Schwann cells, rapid
multiplication of these cells and segregation of the
individual axons by Schwann cell processes
• Smaller axons (≤1 μm)
– remain unmyelinated, are segregated; several
may be surrounded by one Schwann cell, each
within its own pocket
• Large axons (≥1 μm)
– are enclosed singly, one cell per axon per
internodes
• In the CNS, myelin is formed by
oligodendrocytes.
– CNS nerve fibers are not separated by
connective tissue nor are they surrounded by cell
cytoplasm, and specific glial nuclei are not
obviously associated with particular myelinated
fibers.
• In peripheral nerve, the sheath is surrounded by
Schwann cell cytoplasm on the inside and outside
whereas the cytoplasmic tongue in the CNS is
restricted to a small portion of the sheath
 Myelin affects axonal structure
• Transmission of action potentials by saltatory
conduction
• Increase the axonal diameter by inducing
biochemical changes in components of the axonal
cytoskeleton such as neurofilaments
• Signaling mechanisms from myelin or myelin-
forming glia to axons
• Transgenic mice deficient for some of the myelin
proteins.
– necessary for the normal formation, maintenance and
survival of the axons that are ensheathed
 Characteristic composition of myelin
• Separation technique: Homogenization in the media of Low Ionic strength. Large
vesicle size and low intrinsic density.PNS need Vigrous homogenization.
• A lipid bilayer with integral membrane proteins embedded in the bilayer and other
extrinsic proteins attached to one surface or the other by weaker linkages.
• The apposition of the extracellular (Ext.) surfaces of the oligodendrocyte or
Schwann cell membranes to form the intraperiod (IP) line are shown in the upper
part of the figure.
• The apposition of the cytoplasmic (Cyto.) surfaces of the membranes of the
myelin-forming cells to form the major dense (MD) line are shown in the lower part
of the figure.
Central nervous system myelin is enriched in certain lipids.

• Cerebroside (galactosyl ceramide) is the most typical of myelin

• The concentration of cerebroside in brain is directly
proportional to the amount of myelin present
• One-fifth of the total galactolipid in myelin is sulfatide
• UDP-galactose:ceramide galactosyltransferase-null mouse->
NO cerebroside biosynthesis and no sulfatide formation
 Cholesterol and Phospholipid
 On a molar basis, CNS myelin preparations contain cholesterol, phospholipid and
galactolipid in a ratio varying between 4:3:2 and 4:2:2.
 Myelin contains substantially more molecules of cholesterol than any other single lipid.
 Most abundant phospholipid ethanolamine-containing plasmalogen
(glycerophospholipid containing an alkenyl ether bond)
 Lecithin is also a major myelin constituent, and sphingomyelin is a relatively minor
one.
 On the basis of weight the content of galactolipids is comparable
 Cholesterol is enriched on the extracellular face of the myelin membrane, whereas
ethanolamine plasmalogen is asymmetrically localized to the cytoplasmic half of the
bilayer.
 The lipid class composition AND the fatty acid composition of many of the individual
lipids is distinctive in myelin.
PNS & CNS myelin lipids are qualitatively similar
• Quantitative differences.????
• PNS myelin has less cerebroside and sulfatide and
considerably more sphingomyelin than CNS myelin.
• The presence of the LM1 ganglioside,
sialosyl-lactoneotetraosylceramide, as a
characteristic component of myelin in the PNS of
some species.
 Unique proteins of CNS
• The protein composition of CNS myelin is simpler than that of other
brain membranes
• Many other proteins and glycoproteins are present to a lesser extent
• With the exception of MBP, myelin proteins are neither easily
extractable nor soluble in aqueous media.
• Like other membrane proteins, they can be solubilized in sodium
dodecyl sulfate solutions
• The quantitative predominance of two proteins in human CNS
myelin is clear, i.e. MBP and PLP.
– These two proteins are major constituents of all mammalian CNS myelin
membranes and similar proteins are present in myelin membranes of many
lower species.
1. SDS→ separates proteins primarily
according to their molecular weight (Mr for
relative molecular mass, and molecular
weight in kilodaltons, kDa)
2. Electrophoretic migration Vs molecular
weight
3. Apparent molecular weight

Rat
Human
P C
P C

MOG-myelin–oligodendrocyte glycoprotein
CNP-2′,3′-cyclic nucleotide 3′-phosphodiesterase
MAG-myelin-associated glycoprotein
 Proteolipid protein
 Molecular Mass 30,000, migrates anomalously on SDS & gives lower molecular
weight.
 Tetraspan protein (N and C termini in the cytoplasmic end)

 Stabilizes intraperiod line of CNS myelin

 the extracellular loops are found in this region
 abnormal condensation of CNS intraperiod line in PLP knockout mice and
spontaneously occurring PLP mutants
 DM20 (MW 20,000); alternatively spliced isoform of PLP, similar sequence as
PLP except 35 aa deletion form the intracellular domain, Occurs in lower
concentration than PLP
 Similar physical property as PLP
 PLP/DM20: 4-6 mol of FA per mole of protein(palmitate, oleate or stearate), rapid
turnover of FA independent of peptide backbone
 PLP Genetics
The PLP/DM20 gene may have evolved from an ancestral gene
encoding a pore-forming polypeptide (myelin may be involved in ion
movement)
The PLP & DM20 gene is expressed very early in development
DM20 mRNA might have a role in oligodendrocyte migration or
differentiation in addition to a structural role in myelin.
Appears earlier than PLP even before myelin formation in embryos
and in premyelinating oligodendrocytes

 DM20 cannot replace PLP in transgenic mice

 PLP uniquely interacts both with inositol hexakisphosphate a
molecule involved in vesicle transport, and with integrins, modulating
interaction with the extracellular matrix.
 Although PLP and DM20 serve important
functions, they are not essential
A knockout mouse for PLP/DM20 is initially relatively normal with respect to
myelin formation (except for the difference in the intraperiod line spacing), life
span and motor performance
 other proteins or lipids of myelin may contribute to adherence of the extracellular faces of the bilayers
at the intraperiod line

Myelin in the PLP-null mutant is extra sensitive to osmotic shock during

fixation
 PLP does enhance the stability of myelin, possibly by forming a ‘zipper-like structure

Significant axonal degeneration in older PLP/DM20 knockout mice, suggests

that myelin can form in the absence of PLP/DM20 but CNS myelin devoid of

PLP/DM20 cannot sustain normal axonal function

 PLP would be needed for formation of compact, multilamellar myelin
PLP has selective and apparently important
functions in the CNS relative to DM20.
• While the loss of PLP/DM20 is significantly less serious than
expression of mutated or excess PLP/DM20
• Both human patients and genetically engineered or naturally
occurring animal mutants with defects in the PLP gene exhibit
hypomyelination and often early death.
– either abnormal protein that cannot fold correctly or simply increased amounts
of normal PLP which induce an unfolded protein response and are toxic to
oligodendrocytes
 PLP Distribution
• PLP/DM20 expression is highest in oligodendrocytes
– Also expressed in myelinating and nonmyelinating Schwann cells but

do not incorporated into myelin in appreciable amounts.

• DM20 mRNA being expressed more in nonmyelinating Schwann

cells and

• PLP mRNA being expressed more in myelinating Schwann

cells.

• Low levels of DM20 expression have been found in thymus and

heart, again suggesting that this protein has unique functions

unrelated to formation and maintenance of compact myelin
 Myelin basic proteins
• The MBP & EAE
• With dilute acid or salt solutions MBP can be
isolated from Myelin, very soluble in water
after extraction
• MBP genes are highly conserved
• Like PLP gene MBP gene also alternatively
spliced
 MBP Splicing 21,500 kDa

 The
The ratio
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with more
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found
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
InIn immature
immature oligodendrocytes,
oligodendrocytes, the
the MBP
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 However,
However, asas
thethe cell
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from the
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presumably because
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newly translated
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MBP associates
associates rapidly
rapidly with
with membranes
atmembranes at its site of synthesis.
its site of synthesis.
 Axons 2, 5B and 6 are present or absent in four other MBP proteins found in myelin
 The most abundant MBP in human myelin contains exons 1B, 3,4 , 6 and 7 (18.5 kDa)
 In rodent myelin both the 18.5 kDa MBP and a 14 kDa MBP containing exons 1B, 3, 4, 5
and 7
 Two different minor MBPs of approximately 17 kDa exist, which are encoded by exons 1B,
2, 3, 4, 5B and 7 or 1B, 3, 4, 6 and 7 spectively
 MBP
 The MBPs are highly unfolded in solution, with essentially no tertiary
structure
 Microheterogenicity upon electrphoresis in alkaline condition

phosphorylation, loss of the C-terminal arginine, and deamidation,

methylation of an arginine at residue 106
 The rapid turnover of the phosphate groups might influence the close
apposition of the cytoplasmic faces of the membrane

 MBP forms dimers, localized exclusively at the cytoplasmic surface and

stabilizing the major dense line of CNS myelin by interacting

with negatively charged lipids.
 Mutant fail to compact the major dense line
 Enzymes associated with myelin.
 Myelin is metabolically active in synthesis, processing and metabolic
turnover of some of its own components thus a large number of
enzymes have been discovered in myelin
 Play an active role in ion transport with respect not only to maintenance
of its own structure but also to participation in ion buffering near the
axon.
 Myelin and Oligodendrocyte specific Enzymes
 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNP)
 pH 7.2 cholesterol ester hydrolase
 Enzymes that are not myelin-specific but appear to be intrinsic to myelin
 cAMP-stimulated kinase, calcium/calmodulindependent kinase, protein kinase C, a neutral
protease activity and phosphoprotein phosphatases.
 Enzymes associated with myelin.
Steroid-modifying enzymes and cholesterol-esterifying enzymes
UDP-galactose:ceramide galactosyl-transferase
 Enzymes of glycerophospholipid metabolism
Phosphatidyl ethanolamine synthesis from diacyl-glycerol and
ethanolamine
 Myelin may have an active role in ion transport in and out of the axon
 Enzymes involved in ion transport
 Membrane bound carbonic anhydrase
removal of carbonic acid from metabolically active axons
 5′-nucleotidase
Transport mechanism for adenosine

 Na+, K+-ATPase exists in myelin at low levels.

Neurotransmitter receptors associated with myelin
 Neurotransmitter receptors have been identified on oligodendrocytes and
oligodendrocyte progenitor cells as well as in compact myelin
 Muscarinic acetylcholine (mACh) receptors,
 MuscarOligodendroCytosinic receptors may be involved in
phosphatidylinositol signaling in myelin or
 muscarinic receptor activation alters integrin function in
oligodendrocytes by modulating binding to extracellular matrix
molecules
 α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)
receptors,
 glutamate cytotoxicity in oligodendrocyte progenitor cells

 N-Methyl-D-Aspartate (NMDA) receptors and

 Kainate receptors
Development and Metabolic aspects of Myelin

Large neural pathways become myelinated before they are

completely functional
PNS myelinate first, then the spinal cord, and the brain last

Rats and other nest-building animals are quite helpless at

birth and myelinate predominantly postnatally
Grazing animals such as horses, cows and sheep have
considerably more CNS myelin at birth and a
correspondingly higher level of complex activity
immediately postnatally
 Synthesis of myelin components is very
rapid during deposition of myelin
• Rat CNS undergoes considerable development postnatally
• Maximal rate of oligodendroglial precursor cell proliferation occurs
at 10 days and begins to form myelin postnatally (10-12)
– Maximal rate achieved by 20 days after that myelin
accumulation starts to decline throughout the adult hood
• Myelin accumulates in a 20-day-old rat brain at a rate of about 3.5
mg/day
• Rapid incorporation of radioactive precursors study
– The amount of myelin membrane made by each cell per day is more than
three times the weight of its own perikaryon
Sorting and transport of lipids and proteins
• The biogenesis of myelin sheaths requires the spiraling of numerous
oligodendroglial processes around axons and their tight layering to form compact
myelin.
• Additional modeling of specialized membrane domains with different
composition at the inside and outside of the sheaths and in the paranodal glia–
axon junctions

• PLP is synthesized on membrane-bound polysomes in the perikaryon and

transported in membranous vesicles to the myelin being formed at the end of the
oligodendroglial processes.

• MBP is synthesized on free polysomes, which are actually located in very close
proximity to the newly forming myelin at the end of oligodendroglial processes.
(mRNA transport)
• How to measure ???
Sorting and transport of lipids and proteins
• Proteins that are selectively localized in specialized regions
of the myelin sheath must be sorted and transported by
different mechanisms involving specific sorting signals
• Cell polarization may play a role
• Raft like domains (enriched in cholesterol, glycosphingolipids and
glycosylphosphatidylinositol linked proteins) play an important
role in the trafficking of membrane components and signal
transduction mechanisms
Myelin composition changes during development.

• Myelin composition of immature, rapidly developing brain is different from

adult
• Myelin first produced by oligodendrocytes may represent a transitional
form with properties intermediate between those of mature compact
myelin and the oligodendroglial plasma membrane.
• The content of galactolipids, MBP and PLP in purified myelin INCREASES
whereas phosphatidylcholine and high-molecular-weight proteins
DECREASES rat brain matures
• Fractionation: sub fractions of different densities
– lighter fractions are enriched in multilamellar myelin
– denser fractions contain a large proportion of single membrane
vesicles
 Spontaneous Mutations in Experimental Animals
• Myelin mutants: shiverer, jimpy, quaking and
trembler
• The naturally occurring mouse mutants have
phenotypic differences from null mutants
Spontaneous Mutations in Experimental Animals

• The ultrastructure of PNS myelin is normal in these mutants

• PLP is virtually absent from peripheral myelin and P0

appears to be capable of stabilizing both the intraperiod and
major dense lines of myelin of the PNS in the absence of
MBP
• The shiverer phenotype can be corrected by introducing
normal MBP into transgenic mutant mice, resulting in almost
complete correction of the shivering, early death and failure
of CNS myelin compaction.
 PLP Mutation
• The severity of the myelin deficiency in PLP mutants varies.
• Most PLP mutations are quite severe
• PLP mutation in rumpshaker mice causes a relatively mild
hindlimb shaking phenotype.
• By contrast, there is severe reduction and abnormal structure of
CNS myelin in jimpy mice, along with a profound loss of
oligodendrocytes
• The PLP mutants demonstrate the fact that protein mutations
may be far more severe than simple loss of the protein.
Myelin components exhibit great heterogeneity of
metabolic turnover
• The overall rate of metabolic turnover of myelin is
substantially slower than that of other neural membranes
• Structural lipid components of myelin (cholesterol,
cerebroside and sulfatide) and proteins of compact myelin,
are relatively stable
• The metabolic turnover of individual myelin components is
multiphasic
• The presence of signal transduction systems in myelin
sheaths involve rapid events with half-lives on the order of
minutes.
– Polyphosphatidylinositol
– Phosphate groups on MBP