The myelin–axolemmal complex: biochemical dissection and the role of galactosphingolipids

The myelin–axolemmal complex: biochemical dissection and the

role of galactosphingolipids

Krishna Menon,* Matthew N. Rasband,* Christopher M. Taylor,* Peter Brophy,� Rashmi Bansal*

and Steven E. Pfeiffer*

*Department of Neuroscience, University of Connecticut Medical School, Farmington, Connecticut, USA

�Department of Preclinical Veterinary Sciences, Center for Neuroscience at the University of Edinburgh, Summerhall, Edinburgh, UK

Abstract

Myelin–axolemmal interactions regulate many cellular and

molecular events, including gene expression, oligodendrocyte

survival and ion channel clustering. Here we report the

biochemical fractionation and enrichment of distinct subcel-

lular domains from myelinated nerve fibers. Using antibodies

against proteins found in compact myelin, non-compact

myelin and axolemma, we show that a rigorous procedure

designed to purify myelin also results in the isolation of the

myelin–axolemmal complex, a high-affinity protein complex

consisting of axonal and oligodendroglial components. Fur-

ther, the isolation of distinct subcellular domains from galact-

olipid-deficient mice with disrupted axoglial junctions is altered

in a manner consistent with the delocalization of axolemmal

proteins observed in these animals. These results suggest a

paradigm for identification of proteins involved in neuroglial

signaling.

Keywords: axolemma, axon, juxtaparanode, myelin, oligo-

dendrocyte, paranode.

J. Neurochem. (2003) 87, 995–1009.

Myelin is a complex, metabolically active, multilamellar

membrane that ensheaths axons. Electrically, this sheath

confers high resistance and low capacitance to the axon

membrane, thereby facilitating rapid and energy-efficient

conduction of action potentials. Besides these well known

electrical properties, the unique association between myelin

and axolemma is central to the reciprocal exchange of signals

between oligodendrocytes and neurons that regulate diverse

cellular activities. For example, axons regulate oligodendro-

cyte gene expression, signal transduction, sheath thickness

and survival (Friedrich and Mugnaini 1983; Chakraborty

et al. 1999; Lopresti et al. 2001); radiolabeling studies have

provided evidence for the transfer from axon to myelin of

various molecules, including lipids, polyamines (Toews and

Morell 1981; Alberghina et al. 1982; Linquist et al. 1985;

Ledeen et al. 1992) and metabolic precursors (Chakraborty

et al. 2001). In the case of oligodendrocytes and Schwann

cells, they regulate axon caliber (Aguayo et al. 1979;

Sanchez et al. 1996; Yin et al. 1998), microtubular proper-

ties (Kirkpatrick et al. 2001; Dashiell et al. 2002) and

clustering of ion channels and cell adhesion molecules at and

near nodes of Ranvier (Arroyo and Scherer 2000; Peles and

Salzer 2000; Rasband and Trimmer 2001a; Girault and Peles

2002). Although some of the cellular and molecular mech-

anisms for these associations and interactions have been

described (Franzen et al. 2001; Charles et al. 2002), the

neuroglial signaling mechanisms and proteins responsible for

these events are largely unknown.

Recent efforts to define the complex community of proteins

involved in the reciprocal subcellular differentiation and

signaling between myelin and axolemma have identified

several adhesive protein complexes that may be key elements.

For example, the integrity of the paranode, formed by the

close apposition of terminating layers of the myelin sheath

and the axonal membrane, is thought to be mediated in part by

Received July 3, 2003; revised manuscript received July 31, 2003;

accepted August 8, 2003.

Address correspondence and reprint requests to Professor Steven E.

Pfeiffer, Department of Neuroscience, University of Connecticut Health

Center, 263 Farmington Avenue, Farmington CT 06030–3401, USA.

E-mail: [email protected]

Abbreviations used: Caspr, contactin-associated protein; CGT, UDP-

galactose-ceramide galactosyltransferase; CNP, 2¢,3¢-cyclic nucleotide

3¢-phosphohydrolase; GFAP, glial fibrillary acidic protein; MAG, mye-

lin-associated glycoprotein; Kv, voltage-gated potassium; MBP, myelin

basic protein; MOG, myelin oligodendrocyte glycoprotein; NaCh, volt-

age-gated sodium channel; NF-140, neurofilament protein-140 kDa; NF-

155, neurofascin-155 kDa; nH2O, nanopure water; NPJ, node–paran-

ode–juxtaparanode; OL, oligodendrocyte; OSP, oligodendrocyte-specific

protein; PB, phosphate buffer; PLP, proteolipid protein; TBS, Tris-buf-

fered saline; WT, wild type.

Journal of Neurochemistry, 2003, 87, 995–1009 doi:10.1046/j.1471-4159.2003.02075.x

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 995–1009 995

a trimeric protein complex that includes the cell adhesion

molecules contactin-associated protein (Caspr) and contactin

in the axon membrane and oligodendroglial neurofascin-

155 kDa (NF-155) (Menegoz et al. 1997; Peles et al. 1997;

Peles and Salzer 2000; Tait et al. 2000; Charles et al. 2002).

When the normal expression and/or localization of these

proteins at paranodes is altered, axoglial junctions fail to form

and ion channel clustering is perturbed, resulting in conduc-

tion deficits (Dupree et al. 1999; Bhat et al. 2001; Boyle

et al. 2001). In contrast to this well understood protein

complex, a direct interaction of the innermost periaxonal

lamellae of compact myelin with the axon has been predicted,

perhaps through the myelin-associated glycoprotein (MAG),

the Nogo-66 receptor, microtubule associated protein 16

(MAP1b) and/or other axolemmal proteins (Franzen et al.

2001; Domeniconi et al. 2002; Marcus et al. 2002).

In order to further define and identify the molecular

interactions responsible for neuroglial signaling, we have

undertaken a biochemical ‘dissection’ and fractionation of

myelin and axolemmal proteins. Traditionally, myelin ‘puri-

fication’ has relied on its high lipid content, rendering it a low-

density membrane that can be separated from other cellular

constituents and membranes by density gradient centrifuga-

tion (Norton and Poduslo 1973; Agrawal et al. 1974; DeVries

et al. 1978; Haley et al. 1981; Huber et al. 1994). Neverthe-

less, gradient centrifugation approaches have been used

effectively to identify subfractions of myelin, especially in

regard to changes in myelin membrane density as a function

of development (Benjamins et al. 1973, 1976; Pereyra et al.

1983; Shimomura et al. 1984) and to detect alterations in

membrane fragility in mutant animals (Jurevics et al. 2003).

We postulated the existence of high-affinity neurogl-

ial interactions that could be isolated based on their

co-purification with myelin membranes. Further, recent

advances in the identification of specific myelin and

axolemmal proteins restricted to discrete subcellular com-

partments of myelinated nerve fibers (Arroyo and Scherer

2000; Peles and Salzer 2000; Pedraza et al. 2001; Rasband

and Trimmer 2001b) has made possible a detailed analysis of

the protein complexes isolated across the sucrose gradient.

Here we show that distinct subcellular domains of

myelinated nerve fibers can be biochemically isolated but

that, despite rigorous efforts to purify myelin to homogen-

eity, high-affinity neuroglial interactions result in co-purifi-

cation of axolemmal proteins. This latter result shows the

existence of a neuroglial protein complex that we have called

the myelin–axolemmal complex.

Materials and methods

Isolation and fractionation of the myelin–axolemmal complex

Brains from 21-day-old mice (8–10 brains, �3.4 g) were rapidly

dissected, frozen on dry ice and stored at ) 80�C. Solutions of 0.32,0.75, 0.85 and 1 M sucrose (10.2, 23.1, 26.0 and 30.1% respectively,

by refractometry) were prepared in 2 mM EGTA, pH 7.5. Brains

were thawed in 0.32 M sucrose (5% w/v homogenate) and serially

disrupted in a glass and stainless steel (clearance 0.0001 inch)

Dounce homogenizers (15 strokes each).

The homogenate (18 mL) was layered over 0.85 M sucrose

(18 mL) in four centrifuge tubes and centrifuged (Gradient I,

Fig. 1a; Beckman SW28 rotor, 140 000 g, 1 h). Following

centrifugation, 13 fractions were collected based on the visual

appearance of material in the gradient (Fig. 1a): an initial 6-mL

fraction (#1), 11 2-mL fractions (#2–12), and a 7-mL fraction (#13).

The corresponding fractions from the four tubes were combined.

The remaining pellets (�160 mg protein) were combined and

(a)

(b) (c) Fig. 1 Myelin–axolemma purification strat-

egy and distribution of major myelin pro-

teins. (a) Characteristics of Gradients I, II

and III. (b, c) Sucrose density profiles and

immunoblots for MBP, PLP and CNP for

Gradients (b) I and (c) II. Note the signifi-

cant presence of major myelin proteins in

the high-density fractions of the gradients.

LD band, low-density band; L dispersion,

light dispersion; H dispersion, heavy

dispersion; A, heavy band A; B, heavy band

B; P, pellet.

996 Krishna Menon et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 995–1009

suspended in 50 mM Tris-HCl, pH 7.5 (final volume �16 mL). The

sucrose percentages (refractometry, w/w) (Fig. 1b) and protein

contents (DC protein assay kit; Bio-Rad, Richmond, CA, USA)

(Table 1) were determined before immunoblot analysis for the major

myelin proteins myelin basic protein (MBP), proteolipid protein

(PLP) and 2¢,3¢-cyclic nucleotide 3¢-phosphohydrolase (CNP)

(Fig. 1b; 5 lg protein per lane; fractions 1–4 had very little proteinand were not analyzed). The remaining material was stored at ) 80�C.Based on appearance and immunoblot analysis (Figs 1a and b),

fractions from the ‘main band’ (fractions 7–9, 11–25% sucrose; high

MBP and PLP concentrations) and ‘dispersed band’ (fractions 10–13,

26–26.2% sucrose; reduced MBP and PLP concentrations) were

combined. The pellet (lowMBP and no detectable PLP) was stored at

) 80�C. The combined fractions 7–13 were diluted to 200 mL with

10 mM EGTA in nanopure water (nH2O; Barnstead, Dubuque, IA,

USA), pH 7.5, homogenized (glass Dounce, 15 strokes) and

centrifuged (Beckman Ti60 rotor, 200 000 g, 20 min). The resulting

pellets were suspended and combined in 120 mL 10 mM EGTA in

nH2O, pH 7.5, homogenized as above, stirred 15 for min and

centrifuged (Beckman JA17 rotor, 35 000 g, 15 min); this procedure

was repeated once. The final pellets were resuspended in a total

volume of 18 mL 0.85 M sucrose and homogenized (glass Dounce).

Next, a single discontinuous sucrose gradient was prepared

[Gradient II: 2 mL 1 M sucrose, 18 mL 0.85 M sucrose pooled

material (above), 18 mL 0.32 M sucrose; Fig. 1a] and centrifuged

(SW28 rotor, 140 000 g, 90 min, 4�C). Fourteen fractions were

collected from the top based on the visual appearance of material in

the gradient (Fig. 1a): one 6-mL fraction (#1), 11 2-mL fractions

(#2–12), one 7–8-mL fraction (#13), a final 3-mL fraction containing

cloudy and white material (#14), and a pellet. The sucrose

percentages (Fig. 1c) and protein contents (Table 1) were determined

before immunoblot analysis for MBP, PLP and CNP (Fig. 1c; 5 lgprotein per lane; fractions 1–7 had very low levels of protein and

were not analyzed). The remaining material was stored at ) 80�C.Based on appearance and immunoblot analysis (Figs 1a and c),

fractions from the ‘main band’ (fractions 8–9, 19.1–24.2% sucrose;

high MBP and PLP concentration) and ‘dispersed band’ (fractions

10–13, 24.8–24.8% sucrose; lower MBP and PLP concentration)

(Fig. 2b) were combined. Fraction 14 (28.2% sucrose; low MBP and

PLP) and the pellet were stored at ) 80�C without further analysis.

The combined fractions 8–13 were diluted to 200 mL with 2 mM

EGTA in nH2O, pH 7.5, homogenized (glass Dounce, 15 strokes)

and centrifuged (Beckman Ti60 rotor, 200 000 g, 20 min). The

resulting pellets were suspended and combined in 120 mL 2 mM

EGTA in nH2O, pH 7.5, homogenized as above (no stirring) and

centrifuged (Beckman JA17 rotor, 35 000 g, 15 min). The pellets

were resuspended and combined in a total volume of 12 mL 0.85 M

sucrose and homogenized (glass Dounce).

Finally, a single discontinuous gradient was prepared in an

UltraClear SW28 rotor tube [Gradient III: 12 mL 0.85 M sucrose

combined fractions 8–13 from Gradient II (above), 12 mL 0.75 M

sucrose and 12 mL 0.32 M sucrose; Fig. 1a] and centrifuged (SW28

rotor, 140 000 g, 16 h, 4�C). Fractions (1 mL) were collected, and

percentage sucrose and protein contents were determined (Figs 2a

and b). Six regions could be identified based on the sucrose gradient

banding pattern and are described further in Results. Pooled or

individual fractions were diluted in two volumes of 2 mM EGTA in

nH2O, pH 7.5, mixed by vortexing, and centrifuged (Beckman

JA17, 35 000 g, 15 min, 4�C). The resulting pellets were suspendedin 50 mM Tris, pH 7.5, with protease inhibitor cocktail (1 mM

phenylmethylsulfonyl fluoride, 10 lg/mL leupeptin, 10 lg/mLaprotinin) and frozen at ) 80�C. All gradient analyses were done

three times, were highly reproducible, and typical examples are

shown in the results section.

Gel electrophoresis and immunoblotting

Samples were solubilized in urea buffer (50 mM Tris-HCl, 5% SDS,

4 M urea, pH 6.8), mixed with sample buffer (Laemmli 1970) and

loaded without boiling. Proteins (5 lg per lane) were electrophoret-ically separated on 12% SDS polyacrylamide gels (for some analyses

Table 1 Summary of protein content in gradient fractions

Material

Gradient I Gradient I I Gradient III

WT CGT-Null %Null/WT WT CGT-Null %Null/WT WT CGT-Null %Null/WT

Homogenate 378 331 88 40 37 92 15 10 69

Low-density band – – – – – – 0.6 0.7 118.4

Main band (MB) 61.4 57.4 93.6 13.2 8.6 65.4 7.2 2.6 36.6

Dispersion (D) 20.7 22.2 107.3 7.1 8.9 125.3 1.8 2.5 139.3

MB + D 82.0 79.6 97.0 20.2 17.5 86.3 – – –

Fraction 14 – – – 9.9 8.3 83.8 – – –

Heavy band A (A) – – – – – – 0.4 0.6 139.0

Heavy band B (B) – – – – – – 1.1 1.9 171.3

MB + D + A + B – – – – – – 10.5 7.6 72.2

Pellet 167.2 166.1 99.3 7.2 5.6 78.7 2.8 2.3 81.3

Supernatant* 41.6 42.3 101.7 5.7 7.5 131.7 – – –

Total protein – – – 37.3 31.4 84.2 13.9 10.6 75.9

Total wet brain weight of starting material: WT ¼ 3.4 g; CGT-null ¼ 3.4 g. All values in the table are expressed in millligrams of protein. Gradient I,

initial brain homogenate; Gradient II, after osmotic shock of MB + D from Gradient I; Gradient III, after osmotic shock of MB + D from Gradient II.

*Amount lost in the supernatant is calculated by subtracting total protein of main band and dispersion at the end of run from the total protein at the

start of the next gradient.

Myelin–axolemmal complex 997


of PLP and MBP, 1 lg per lane and 15% SDS polyacrylamide gels

were used) (Laemmli 1970) and electrophoretically transferred at

100 V for 1 h on to Immobilon-P membranes (Amersham, Piscata-

way, NJ, USA). Blots were blockedwith a solution of 5% non-fat milk

in Tris-buffered saline (TBS; 25 mM Tris, 2.6 mM KCl, 0.14 M NaCl,

pH 8.0) for 1 h, incubated with primary antibody, washed three times

(TBS, 0.2% Tween 20), incubated with the appropriate secondary

antibody for 20 min, washed, and visualized by enhanced chemilu-

minescence using ECL or ECL-Plus on Hypermax film (Amersham).

Blots were reprobed after stripping of antibody using a 100-mM

glycine solution at pH 2. Relative band intensities were determined by

scanning film images within the linear ranges of grain density

followed by analyses of mean band densities using NIH Image (US

National Institutes of Health, Bethesda, MD, USA). These band

densities were then used to determine relative ‘concentrations’ of

specific proteins in each fraction by determining the ratio of the band

densities to the total protein content (a form of relative specific

activity), allowing one to adjust for differences in the amounts of

material among the fractions. All analyseswere done three times, were

highly reproducible, and typical examples are shown in the results

section.

Antibodies

Antibodies used were against myelin oligodendrocyte glycoprotein

(MOG; mouse monoclonal, IgG1, 1 : 3000; C. Linington, Munich,

Germany), MAG (rabbit polyclonal 1 : 10 000; J. Roder, Toronto,

ON, Canada), CNP (rabbit polyclonal 1 : 10 000; Sternberger

Biochemicals, Lutherville, MD, USA), claudin-11/oligodendro-

cyte-specific protein (OSP; rabbit polyclonal 1 : 5000; J.

Bornstein, Los Angeles, CA, USA), MBP isoforms (rabbit

polyclonal 1 : 10 000; Barbarese et al. 1977), PLP (AA3, rat

monclonal 1 : 10 000; M. Lees, Boston, MA, USA), Fyn (mouse

monoclonal 1 : 2000, IgG1; Transduction Laboratories, Lexington,

KY, USA), Caspr-1 (rabbit polyclonal 1 : 3000, E. Peles, Rehovot,

Israel; IgM mouse monoclonal, Rasband and Trimmer 2001a),

NF-155 (rabbit polyclonal 1 : 1000; P. Brophy, Edinburgh, UK),

glial fibrillary acidic protein (GFAP; rabbit polyclonal 1 : 3000;

DAKO Corporation, Carpinteria, CA, USA), beta-tubulin III (b-tub-3) (mouse monoclonal, IgG2b, 1 : 2000; Sigma Chemicals, St

Louis, MO, USA), sodium channel (pan mouse monoclonal, IgG1,

1 : 500; Rasband et al. 2001), the voltage-gated potassium (Kv)

channel subunits Kv1.2 (mouse monoclonal IgG 1 : 600;

Bekele-Arcuri et al. 1996) and Kvb2 (mouse monclonal, IgG

(a)

(b)

(c)

Fig. 2 Characterization of the myelin–axo-

lemmal fractions across Gradient III. (a)

Analysis of total proteins and density (%

sucrose) for fractions from WT (+/+) and

CGT-null (–/–) mouse brain myelin. LDB,

low-density band; A, heavy band A; B,

heavy band B; n ¼ wt, d ¼ null, j ¼ %

sucrose (b) Representative immunoblots

showing the distribution of various myelin

and node–paranode–juxtaparanode (NPJ)

molecules in different fractions (5 lg pro-

tein/lane) across the gradient. Note the shift

in NPJ molecules into the main band (frac-

tions 14–17) from CGT-null compared with

WT myelin, and the virtual absence of

GFAP and NF-140. Tub, tubilin (c) Trans-

mission electron micrographs of material

from the main band (fraction 14), light dis-

persion (fraction 25) and heavy band B

(fractions 34–37). The main band is en-

riched in multilamellar structures, whereas

heavy band B is composed largely of uni-

lamellar structures. Scale bar 1 lm.



1 : 400; Bekele-Arcuri et al. 1996), Caspr-2 (mouse monoclonal

IgG1, culture supernatant; J. Trimmer, State University of New York

(SUNY), Stony Brook, New York, USA), Post synaptic density

(PSD)-95 (mouse monoclonal IgG, tissue culture supernate;

Rasband et al. 2002), and neurofilament protein 140 kDa (NF-

140; rabbit polyclonal, 1 : 1000; Chemicon Laboratory, Temecula,

CA, USA). Species-specific secondary antibodies were generally

used at 1 : 3000, but reduced to 1 : 10 000 for the detection of anti-

MAG, anti-MBP and anti-PLP binding.

Immunohistochemistry

Optic nerves from postnatal day 22 (P22) and P35 wild-type (WT)

and UDP-galactose-ceramide galactosyltransferase (CGT)–/– mice

were fixed [4% paraformaldehyde, 0.1 M phosphate buffer (PB),

pH 7.2, 30 min], equilibrated (20% sucrose in 0.1 M PB), and

frozen at )30�C in Tissue-Tek OCT mounting medium (Miles,

Elkhart, IL, USA). Sections (5 lm) were placed in 0.1 M PB, spread

on gelatin-coated coverslips, air dried, permeabilized [2 h, 0.1 M

PB, pH 7.4, 0.3% Triton X-100, 10% goat serum (PBTGS)],

incubated with primary antibodies overnight at room temperature,

washed (three times, 5 min each in PBTGS), incubated for 1 h with

secondary antibodies [goat anti-IgG1 mouse antibody Alexa 350

(blue, 1 : 1000) or goat anti-rabbit antibody Alexa 594 (red,

1 : 1000) (Molecular Probes, Eugene, OR, USA); or goat anti-IgM

mouse antibody FITC (green, 1 : 500) (Sigma)], washed, air-dried

and mounted on slides with antifade mounting medium. Digital

images were collected on a Zeiss Axioskop 2 fluorescence

microscope fitted with an Axiocam color camera (Carl Zeiss

Microimaging, Thornwood, NY, USA).

Immunoprecipitation

Immunoprecipitation reactions were performed essentially as des-

cribed in Rasband et al. (2002). For each reaction, 500 lg main

band myelin was solubilized in 0.5 mL lysis buffer for 1 h at 4�C.Detergent extracts were collected by centrifugation at 16 000 g for

30 min, and then incubated with 1–5 lg purified antibody for 2 h.

The detergent-insoluble material was kept for later analysis. Some

50 lg protein A or G conjugated to agarose or sepharose (50% v/v

slurry) was added to the antibody/myelin extract and then incubated

for an additional 45 min. The bead–antibody–antigen complex was

then collected by a short centrifugation to pellet out the complex. An

aliquot of the remaining antigen-depleted supernatant was kept for

later analysis. The beads were then washed seven times with lysis

buffer, pelleted between each wash by centrifugation, and the

immunoprecipitation products eluted from the beads by addition of

200 lL reducing sample buffer and boiling.

Transmission electron microscopy of myelin fractions

Myelin fractions were resuspended in fixative solution (either 3%

paraformaldehyde/0.1% glutaraldehyde or 2.5% glutaraldehyde in

0.1 M sodium cacodylate buffer, pH 7.4) and centrifuged (SW55

rotor, 45 000 g, 20 min). Fixation was continued on ice for 1–2 h.

The fixative was removed and the pellets were rinsed and stored at

4�C in 0.1 M cacodylate buffer with or without 1% paraformalde-

hyde. Samples for morphological analysis were postfixed (1%

osmium tetroxide/0.8% potassium ferricyanide in 0.1 M cacodylate

buffer, pH 7.4, 1 h, room temperature), rinsed in distilled water and

stained (0.5% aqueous uranyl acetate, 1 h). After dehydration in

ethanol solutions, the samples were infiltrated with propylene oxide/

Polybed epoxy resin mixtures (Polysciences, Warrington, PA, USA),

embedded in Polybed (Polysciences), polymerized at 60�C, cut witha diamond knife, collected on bare or formvar-coated copper

specimen grids, stained with uranyl acetate and lead citrate, and

examined in a transmission electron microscope.

Results

Myelin has several unique properties that are essential to its

function. First, it is particularly lipid rich, which contributes

to the passive electrical properties of the myelinated nerve

fiber. Second, it is closely associated with the axolemmal

membrane through adhesive protein complexes thought to

play important roles in establishing discrete myelin–axolem-

mal membrane domains. Third, it is metabolically active,

such that significant bidirectional signaling occurs between

myelin and the axon. To investigate the molecular organiza-

tion of this signaling and to further define the adhesive protein

complexes that exist between myelin and the axolemma, we

developed an enrichment and biochemical fractionation

strategy for these structures. Subsequent to the separation of

myelin–axolemmal membrane compartments, we used highly

specific antibodies against a variety of compact myelin, non-

compact myelin and axolemmal proteins to investigate the

molecular organization of myelin–axolemmal interactions.

Myelin and its associated axolemmal membranes form

high-affinity complexes

P21 mouse crude brain membranes were fractionated by a

sucrose density gradient centrifugation approach designed to

enrich for myelin proteins. Two preliminary gradient steps

(Fig. 1a, Gradients I and II; Table 1) yielded an initial

enrichment for myelin proteins, as indicated by immunore-

activity for MBP, PLP and CNP (Figs 1b and c). Consistent

with previously reported purification strategies, these pro-

teins were highly enriched in the lighter, low-density

fractions 7–9. However, our analysis showed that there was

also a significant amount of these myelin proteins present in

the heavier fractions (10–13) and in the pellet (Figs 1b and

c). Traditionally, fractions 7–9 have been kept as ‘pure

myelin’, whereas fractions 10–13 and the pellet have been

discarded. We hypothesized that high-affinity interactions

between the myelin membrane and the denser axolemma

might account for the sedimentation of myelin membrane

into these heavier fractions. Conversely, if these high-affinity

interactions exist, axolemmal proteins should also be present

in the low-density myelin fractions.

To test this hypothesis, fractions 8–13 from Gradient II

were combined in 0.85 M sucrose, floated into a shallow

sucrose gradient (Fig. 1a, Gradient III; Fig. 2a; Table 1) and

analyzed using an array of antibodies specific for compact

and non-compact myelin proteins, and axolemmal proteins.



After centrifugation to equilibrium, seven regions were

identified based on the visually distinct banding pattern

(Table 2): (1) aggregated material of very low density (low-

density band); (2) major band containing the majority of the

low-density myelin membrane (main band); (3 and 4) two

visually distinct, broad bands with low protein concentra-

tions [light and heavy dispersions]; (5 and 6) visually distinct

bands of higher density (heavy bands A and B); and (7) a

pellet.

Immunoblotting of the resulting fractions was then carried

out for known myelin and axolemmal proteins (Fig. 2b;

parallel studies carried out on material obtained from CGT-

null mice (–/–) are discussed below). Consistent with the

hypothesis that high-affinity myelin–axolemmal interactions

exist, both myelin and axolemmal proteins were found over a

wide range of densities spanning the entire gradient. How-

ever, astrocytic GFAP and axonal NF-140 were not detected.

Electron microscopic examination of representative fractions

demonstrated significant differences between the lowest and

highest density fractions (e.g. main band fraction 14 vs. heavy

band B; Fig. 2c), which were enriched in multilamellar and

unilamellar membrane structures respectively, whereas the

dispersion fractions were a mixture of the two. We therefore

conclude that the procedure to enrich myelin also results in

the co-purification of axolemma, suggesting the existence of

high-affinity interactions with myelin.

Specific myelin and axolemmal proteins

are differentially enriched

In order to assess the degrees of enrichment achieved for

myelin proteins across the gradient profile, we compared

their concentrations in brain homogenate with those in

selected fractions (Fig. 3a). Consistent with the high abun-

dance of myelin proteins in the lighter-density fractions, the

compact myelin protein MBP-17 was optimally enriched

(�35-fold) in the main band; however, even in the heaviest

fractions there was significant enrichment (�10-fold in heavyband B). A similar pattern of enrichment was observed for

the compact myelin protein PLP (data not shown). In

contrast, the enrichments for the inner lamellar myelin

protein MAG, the non-compact myelin protein CNP and the

junctional protein OSP [claudin-11 (Bronstein et al. 1996;

Morita et al. 1999); data not shown] were distinctly different

from that seen for compact myelin proteins, with all three

Table 2 Characteristics of major fractions from gradient III

Fractions Name

Density

(mg/mL)

Sucrose

(%, w/w)

10–13 Low-density band 1.3540 14.0

14–17 Main band 1.3665 21.6

18–15 Light dispersion 1.3695 23.3

26–29 Heavy dispersion 1.3700 23.7

30–33 Heavy band A 1.3705 24.1

34–37 Heavy band B 1.3725 25.0

Pellet Pellet 1.3725 > 25.0

(a)

(b)

Fig. 3 Relative enrichment of selected myelin and NPJ proteins. (a)

Enrichment of selected proteins in the main band, light dispersion, and

heavy bands A and B compared with the original brain homogenate

(H). Data from two independent experiments are shown. Note that

there is a strong enrichment for the compact myelin protein MBP,

especially in the main band, but a uniform enrichment across the

gradient for the myelin inner lamellar protein MAG and the myelin loop

protein CNP. In contrast, there is an enrichment in the heavy bands for

the axolemmal proteins NaCh, Caspr-1 and Kv1.2, whereas PSD-95 is

de-enriched. The astrocyte marker GFAP is virtually absent in all the

fractions. (b) Immunoprecipitation from fraction 14 main band myelin

by anti-Kv1.2 antibody and identification of the interacting partners

Kvb2 and PSD-95 by immunoblotting. IP, immunoprecipitation; depl

supe, depleted supernatant from the IP reaction; insol. pellet, deter-

gent-insoluble pellet. The total protein loaded on the gel is the equiv-

alent of 50 lg of starting material.



showing a uniform enrichment of 8–10-fold across the

gradient.

Immunofluorescence microscopy has shown that a striking

localization of axolemmal proteins occurs at and near the

interface between the paranodal loops of myelin and the

axon, exemplified by clustering of voltage-gated sodium

channels (NaChs) at the node of Ranvier, Caspr-1 and

contactin at the paranode, and Kv channels at the juxtapar-

anode (Figs 4a and c) (reviewed in Rasband and Trimmer

2001b). These proteins had inverse patterns of enrichment

compared with that for the compact myelin proteins, with the

highest degree of enrichment in the heavier fractions

(Fig. 3a). The pattern of enrichment of NF-155, an oligo-

dendrocyte protein localized at the paranodes (Tait et al.

2000), was similar to that for the axolemmal proteins (data

not shown), suggesting the existence of high-affinity inter-

actions between NF-155 and axonal binding partners.

In contrast to these axolemmal proteins, PSD-95, a PDZ-

domain scaffolding protein found at synapses (Hsueh et al.

1997) and juxtaparanodes (Baba et al. 1999), was dramat-

ically de-enriched in the main band, and to a lesser extent in

the heaviest fractions, compared with homogenate (Fig. 3a).

Because PSD-95 co-localizes with Kv1 channels at the

juxtaparanodal junction in immunohistochemical studies

(Rasband et al. 2002), we postulated that the PSD-95 present

in the main band was due to interaction with Kv1 channels

which, in turn, suggests the presence of juxtaparanodal

axolemmal membrane. Immunoprecipitation of the Kv1.2

potassium channel a-subunits from main band (fraction 15)

resulted in co-immunoprecipitation of all detectable Kvb2,and more than half of the PSD-95 (Fig. 3b). This result is in

contrast to previous reports (Rasband et al. 2002) showing

that the Kv1.2–PSD-95 protein complex immunoprecipitated

from whole brain contains only a small fraction of the total

pool of PSD-95 and Kv1.2. Thus we conclude that the PSD-

95 detected in the main band corresponds to the pool of PSD-

95 that interacts with Kv1 channels at juxtaparanodes, and

that the axolemmal proteins found in the main band derive

from membrane near sites of axoglial contact.

Finally, the astrocytic proteins GFAP and connexin 43

were examined as controls for the presence of astrocytic

cytoskeletal and membrane proteins respectively. GFAP was

almost entirely removed from the membrane fractions

(Fig. 3a). The NMDA receptor was studied as a marker for

the inclusion of synaptic membranes; in spite of its presence

in oligodendrocytes, it was strongly de-enriched in this

material (data not shown). Therefore, there appears to be

little inclusion within Gradient III of astrocytic cytoskeletal

or synaptic membranes.

Disruption of the myelin–axolemmal protein complex

Because our analysis of the proteins isolated across the

sucrose gradient suggests the existence of specific high-

affinity interactions between the myelin and axolemmal

membrane, we sought to disrupt this interaction both

biochemically and by studying genetic mutants with altered

axoglial contact. We postulated that disruption of these

interactions would result in a redistribution of axolemmal

proteins into different regions of the sucrose gradient.

Therefore, main band, heavy bands A and B and the pellet

were treated with 1.0 M salt for 15 min with stirring and

centrifuged again on Gradient III; this treatment to disrupt

ionic interactions did not disrupt the interactions between

myelin and axolemma (data not shown).

We then compared and quantified the fractionation of

myelin from WT and mutant mice lacking the enzyme CGT

(CGT-null), an enzyme required for production of galacto-

cerebroside and sulfatide. Phenotypically, CGT-null mice

have shortened life-spans, display severe tremors and have

conduction deficits (Coetzee et al. 1996). CGT-null mice

lack the transverse bands normally found at paranodal

axoglial junctions, resulting in the eversion of paranodal

loops away from the axon and the redistribution of ion

channels into regions from which they are normally excluded

(e.g. Kv1 channels redistribute into paranodal zones). The

delocalization of axolemmal proteins is shown explicitly by

immunofluorescence microscopy of optic nerve sections

(Fig. 4). Whereas WT mice (Figs 4a and c) normally express

clustered NaChs (blue) at nodes of Ranvier, Caspr-1 (green)

at paranodes and Kv1.2 (red) at juxtaparanodes, CGT-null

mice (Figs 4b and d) have little or no clustered Caspr-1, and

aberrantly clustered NaChs and Kv1 channels. Thus, CGT-

null mice represent an ideal model to further test the

(a) (b)

(c) (d)

Fig. 4 Triple immuno-labeling of optic nerve. (a, b) P21 and (c, d) P35

from (a, c) WT and (b, d) CGT-null mouse optic nerve, using anti-

bodies against NaCh (blue), caspr-1 (green) and Kv1.2 (red). Note the

discrete organization of NPJ proteins in WT, but the high degree of

disorganization in CGT-null. Scale bar 10 lm.



hypothesis that the presence of the axolemmal proteins in the

main band by immunoblot (Fig. 2b) is due to high-affinity

interactions between the myelin and axon.

Myelin proteins derived from P21 CGT-null (–/–) mice

had a similar fractionation pattern across Gradients I and II

to age-matched WT (+/+) animals (Figs 1b and c). How-

ever, the ratios of total protein (CGT-null/WT) showed

some small differences across the gradients (Table 1). More

significant differences were identified in the total protein

content and fractionation profiles of these proteins in

Gradient III (Fig. 2a). Specifically, the total yield of protein

from CGT-null mouse brains was only �40% of that of

WT, and a larger proportion of the CGT-null membranes

sedimented to higher densities. Thus, the ratio of the total

protein content was increased in the dispersion and heavy

bands A and B to 139, 139 and 171% respectively. In stark

contrast to this increase in the heavier fractions, the ratio of

protein in the main band was only 37% (Table 1). Similar

studies with P35 WT and CGT-null brain produced similar

results (data not shown). An interesting study by Jurevics

et al. (2003) noted a similar reduction in myelin yield

compared with WT mice from PLPnull mice, apparently due

to the enhanced fragmentation, and thus impaired sedimen-

tation at 20 000 g, of the mutant myelin during the initial

homogenization. This material could be recovered by

centrifuging the 20 000 g supernatant fraction at

100 000 g. In the present case, this situation was avoided

by loading the initial homogenates directly on to the

sucrose density gradients.

Patterns of fractionation of related groups of proteins

We next immunoblotted each fraction from the gradient

(Fig. 2b) using antibodies against compact and non-compact

myelin proteins, as well as axolemmal proteins. The

antibodies used included those against proteins found in

discrete subcellular domains associated with the node of

Ranvier. Specifically, nodal (NaChs), paranodal (Caspr-1,

NF-155) and juxtaparanodal proteins (Kv1.1, Kv1.2, Kvb2,Caspr2 and PSD-95) were tested. To better identify differ-

ences among separation patterns of the proteins across the

gradient, we quantified both the concentration (immunore-

activity per microgram protein) and the total amount of each

protein. The data indicate that the proteins can be divided

into three distinct classes that correlate well with their

presence in compact myelin (Fig. 5), non-compact myelin

(Fig. 6) or axolemma (Fig. 7).

Compact myelin proteins

We found that the major compact myelin proteins (MBPs,

PLP and DM20) were detected throughout the gradient

(Figs 2b and 5). In the WT animals the total amounts were

strongly enriched in the main band (Fig. 5). In contrast,

CGT-null mice had significantly less of the total amount of

major compact myelin proteins in the main band.

The concentrations across the gradient of these proteins

were similar for WT and CGT-null. MBPs were highest in the

main band and decreased gradually through progressively

denser fractions of the gradient. The concentrations of PLP

and DM20 showed a bimodal distribution centered in the

main band and in the heavy dispersion. There were

differences between WT and CGT-null with regard to the

amount of certain proteins per unit of myelin (seen as the

areas under the curves); for example, the amounts of

MBP14 and MBP18.5 were somewhat increased and

decreased respectively.

Non-compact myelin proteins

In contrast to the fractionation patterns seen for the compact

myelin proteins, the pattern for non-compact myelin proteins

associated with cytoplasm-containing paranodal loops or the

inner or outer myelin lamellae (i.e. CNP, MAG, MOG, OSP,

Fyn and NF-155) were different in both WT and CGT-null

material. The total amounts of each of these proteins were

Fig. 5 Total amounts (left panel) and concentrations (right panel) of

the compact myelin proteins MBP and PLP across the gradient in

material from WT (e) and CGT-null (j) mice. The bulk of both MBP

and PLP is found in the main band. LDB, low-density band; A, heavy

band A; B, heavy band B; P, pellet.



distributed bimodally, with significant amounts present not

only in the main band, but also in heavy bands A and B and

the pellet (Fig. 6). Two exceptions included a relatively

reduced level of OSP in the WT pellet, and the dramatic loss

of NF-155 from the CGT-null main band (see below). The

presence of large quantities of these proteins in the heaviest

fractions strongly suggests that these non-compact myelin

proteins are intimately associated with membrane at or near

sites of axoglial interaction.

In both WT and CGT-null mice the concentrations of the

non-compact myelin proteins CNP, MAG, MOG and OSP

remained nearly constant across the gradient (Fig. 6), except

for a sharp reduction in MOG and OSP in the heaviest

fractions. The concentrations of both Fyn and NF-155

gradually increased across the gradient for both WT and

CGT-null, with significant amounts present in the pellet

fraction from WT animals, but a sharp drop in Fyn in the

CGT-null pellet. Particularly worthy of note, the concentra-

tion of NF-155 was dramatically reduced across the entire

gradient to �30% of WT. As NF-155 is the oligodendroglial

component of the tripartite adhesive complex at the paranode

(Tait et al. 2000; Charles et al. 2002), these results suggest

that the phenotype of the CGT-null may be related specif-

ically to the loss of NF-155.

Axolemmal proteins

Finally, immunoblotting with antibodies against proteins

localized at the node of Ranvier, the paranode or the

juxtaparanode revealed a third pattern of total protein

fractionation. Specifically, NaChs, Caspr-1, contactin,

Kv1.1 and Kv1.2 potassium channel a-subunits (Kv1.1 not

shown), Caspr2, Kvb2 and PSD-95 were all present in the


myelin proteins from the outer lamella (MOG) and periaxonal lamella

(MAG), compact and non-compact myelin (CNP, OSP), and paranodal

(NF-155) regions across the gradient from WT (e) and CGT-null (j)

mice. Note the level of NF-155 in the heavy fractions from WT mice

and its dramatic reduction in CGT-null mice. The signaling molecule

Fyn exhibits a pattern similar to that of NPJ molecules and CNP,

whereas MAG is distributed equally across the gradient. LDB, low-

density band; A, heavy band A; B, heavy band B; P, pellet.


nodal (NaCh), paranodal (Caspr-1) and juxtaparanodal proteins

(Caspr-2, Kv1.2, Kvb2 and PSD-95) across the gradient in material

from WT (e) and CGT-null (j) mice. In the WT, the total amounts and

concentrations of all the axolemmal proteins belonging to the NPJ are

enriched in the denser fractions. In the CGT-null, these NPJ proteins

are shifted towards less dense fractions. LDB, low-density band; A,

heavy band A; B, heavy band B; P, pellet.



largest amounts in the heaviest fractions of the gradient in

both CGT-null and WT animals (Figs 2 and 7). However,

there was also a clear subset of the total pool of these proteins

that co-fractionated in the main band with the myelin

proteins, again suggesting the existence of a protein complex

that results in co-purification of axolemma with myelin

proteins. It is interesting to note that the profiles of the total

amounts of both NF-155 and Fyn resemble more closely

those of the ‘axolemmal’ proteins rather than those observed

for the compact or non-compact myelin proteins. Because

NF-155 is a component of the paranodal protein complex it

seems logical that high-affinity interactions will cause this

protein to be co-purified with axonal Caspr-1. In contrast,

Fyn has been described previously as a myelin protein but

without any known high-affinity interactions with the

axolemma or axolemmal proteins (Kramer et al. 1999).

Based on these results, the assignment of Fyn as a ‘myelin’

protein may need to be re-examined.

The concentrations for each axolemmal antigen increased

across the gradient, with the highest levels found in heavier

fractions (Fig. 7). The concentration of the prototypical

nodal protein NaCh was bimodal, with a significant

presence in the lighter-density fractions. This observation

is particularly interesting given that several cell adhesion

molecules (e.g. Navb1, Neurofascin-186 and NrCAM) may

interact with NaChs and result in co-fractionation with

lighter myelin membranes through binding to oligoden-

droglial partners, whereas NaChs associated with heavier

fractions may be recruited to these densities through

associations with cytoskeletal elements. Supporting the idea

that groups of proteins form macromolecular complexes,

the concentration profiles for the paranodal proteins Caspr-1

(Fig. 7) and oligodendroglial NF-155 (Fig. 6), and for the

juxtaparanodal proteins Kv1.2, Kvb2, Caspr-2 and PSD-95

(Fig. 7) respectively, have very similar concentration pro-

files across the gradient.

In strong support of the hypothesis of high-affinity

myelin–axolemmal interactions, a close comparison be-

tween the CGT-null and WT concentration profiles reveals a

clear shift in the fractionation of these proteins toward

lighter sucrose densities in the CGT-null mouse. For

example, the concentrations of both Caspr-1 and Caspr-2,

paranodal and juxtaparanodal cell adhesion molecules

respectively, were dramatically increased and shifted into

the main band. This shift toward fractions enriched in

myelin proteins is consistent with the delocalization of

Caspr-1 and Caspr-2 that has been described previously by

immunohistochemistry (see also Fig. 4). Finally, the shift

was also observed for both NaChs and Kv1 channels, a

result that is consistent with the observed delocalization of

ion channels in the CGT-null mouse (Figs 4b and d). These

results show that the fractionation of mutant myelin results

in an exaggerated concentration of axolemmal proteins in

the lighter fractions and reveals the existence of intrinsic

myelin–axolemmal protein–protein interactions that are not

eliminated by genetic disruption of paranodal axoglial

junctions.

Discussion

A central goal of the present study was to investigate the

interactions between myelin and the axonal membrane in

order to understand the molecular basis of critical signals that

are passed bidirectionally between these two structures. To

this end, we have achieved a density-based fractionation of

myelin and axonal membranes that correlates well with the

discrete membrane compartments observed in myelinated

nerve fibers in situ by immunohistochemistry and electron

microscopy (Bartsch et al. 1989; Brunner et al. 1989; Trapp

et al. 1989; Rasband and Trimmer 2001b; Girault and Peles

2002). A key observation is the presence of specific, high-

affinity interactions between molecules of the myelin sheath

and the axolemmal membrane. These interactions were then

extended to an analysis of myelin from CGT-null animals

that lack the major myelin glycosphingolipids galactocer-

ebroside and sulfatide. By comparing mutant with WT brain,

we have identified a redistribution of the axolemmal protein

profile across the density gradient consistent with the

disruption of the highly ordered axolemmal protein domains

observed by immunohistochemistry in these mutants at and

near nodes of Ranvier; nevertheless, even in these mutant

animals, high-affinity interactions persist. We propose that

what is isolated is a physiological, interacting unit we have

called the myelin–axolemmal complex.

Can one actually ‘purify’ myelin?

How do the results presented here differ from previous

efforts to fractionate and purify myelin? The low-density

main band observed in these studies has traditionally been

taken as purified myelin (Norton and Poduslo 1973).

However, these studies suffered from the lack of available

specific axolemmal markers. A striking result from our

experiments is that we find in this main band low but

significant amounts of ion channels and other axolemmal

proteins that are localized in situ at or near the node of

Ranvier and, conversely, low but significant amounts of

myelin proteins in the heavier fractions. These results are

consistent with extensive previous efforts to purify

axolemmal proteins (Agrawal et al. 1974; DeVries 1976;

Fujimoto et al. 1976; DeVries et al. 1978). This reciprocal

distribution is not simply due to poor fractionation, because

essentially all GFAP and NF-140 immunoreactivity was

removed from the gradient. We propose that the most likely

explanation for this co-purification is the existence of high-

affinity interactions between myelin membrane proteins and

axolemmal proteins, resulting in the attachment of small

fragments of axolemma to myelin membrane fragments.

These interactions survive rigorous homogenization, sucrose



density centrifugation, and high-salt shock to disrupt ionic

interactions. Thus, we conclude that it is difficult, if not

impossible, to purify myelin membrane per se to homo-

geneity.

Patterns of protein distribution as a function of

membrane density

Our biochemical fractionation identifies three distinct

patterns of protein isolation that include compact myelin

proteins, non-compact myelin proteins and axolemmal

proteins. In general, the distributions of the various

myelin proteins over the gradient reflect previous observa-

tions at the level of both cryo-electron and light microscopy

on the subcellular localization of these proteins in the myelin

sheath (Bartsch et al. 1989; Sternberger et al. 1979; Brunner

et al. 1989; Trapp et al. 1989). For example, these structural

studies identify MBP throughout the compact myelin,

whereas MAG is localized to the inner periaxonal lamella.

The observed differential patterns of enrichment for MBP

(main band) and MAG (main band and heavy bands) are

consistent with these subcellular distributions.

NF-155, a glial protein localized at paranodes (Tait et al.

2000), is enriched in both the main band and in the heaviest

fractions in a manner similar to that of Caspr1. These results

are consistent with the idea that NF-155, Caspr and contactin

form a tripartite cell adhesion complex at the paranodal

axoglial junction (Peles and Salzer 2000; Rios et al. 2000;

Charles et al. 2002). Thus, the interaction of NF-155, Caspr1

and contactin appears to be sufficient to recruit NF-155 into

denser membrane fractions.

On the other hand, axolemmal Kv1 channels are localized

in situ at the juxtaparanode (Wang et al. 1993; Rasband and

Shrager 2000; Rasband and Trimmer 2001b). A plausible

mechanism for this localization, and for their pattern of

fractionation on our density gradients, is based on the

concept that Kv1 channels can be recruited to the main band

fractions through their interaction with Caspr2 (Poliak et al.

1999). This hypothesis is made even more tenable by the

recent identification of a glial cell adhesion molecule,

TAG-1, that is also clustered at juxtaparanodes (Traka et al.

2002). It will be interesting to learn whether TAG-1 interacts

directly with Caspr2, or whether it shows a fractionation

profile across the sucrose gradient similar to that of Caspr2.

Our results suggest that other unidentified protein–protein

interactions exist that foster the recruitment of axolemma into

the main band. Future experiments utilizing proteomic

technology should elucidate the full complement of proteins

comprising the myelin–axolemmal complex.

A model for the myelin–axolemmal complex

Based on the data presented here, we propose the following

model (Fig. 8): homogenization of myelinated nerve fibers

generates a continuum of membranes that can be separated

by flotation on sucrose density gradients. These membranes

(a)

(b)

(c)

Fig. 8 Model for the myelin–axolemmal complex. (a) Schematic rep-

resentation of the organization of compact multilamellar myelin and

nodal, paranodal, juxtaparanodal and internodal domains along the

axon. (b) Distribution of material after equilibrium centrifugation on a

three-step discontinuous sucrose (center). The model proposes that

the distribution of material on the gradient results in significant part

from varying ratios of myelin and axolemmal membranes joined by

high-affinity interactions (schematic, left). At low densities, there are

high levels of multilamellar membrane in the main band with a relat-

ively small proportion of attached axolemmal membrane, whereas at

high densities there is a preponderance of unilamellar membranes

with a relatively high proportion of attached axolemmal membrane

(electron micrographs, right). LDB, low-density band; MB, main band;

LD, light dispersion; HD, heavy dispersion; A, heavy band A; B, heavy

band B. (c) A model showing the loss of localization of nodal, para-

nodal and juxtaparanodal proteins in the CGT-null compared with the

WT mouse. Colors denote axolemmal proteins as follows: green,

paranodal; red, juxtaparanodal; blue, nodal.



are derived from distinct domains, and contain specific

proteins intrinsic to the functions of these compartments

(Fig. 8a). The membranes vary in density owing to differ-

ences in lipid/protein ratios among domains, and in the high-

affinity interactions between myelin and axolemma resulting

in the retention of varying amounts of higher-density

attached axolemmal fragments (Fig. 8b). Thus, at the lightest

densities on the gradient there is a high degree of enrichment

for multilamellar, compact myelin; however, small amounts

of axolemmal proteins do co-purify with myelin. At

increasing densities, the preponderance of unilamellar mater-

ial increases with an accompanying increase in the detection

of axolemmal and non-compact myelin and myelin ‘loop’-

related proteins. Thus, we propose that these unilamellar

structures arise from denser myelin ‘loop’ and axonal

membranes. Finally, in CGT-null animals, although there is

a smaller total amount of main band material compared with

WT, nevertheless there is an increase in both the amount and

concentrations of axolemmal proteins in the lower-density

fractions (Figs 2, 5, 6 and 7). The model suggests that this

may be due to two factors (Fig. 8c): first, the absence of

GalC and sulfatide results in a loss of localization mecha-

nisms, thereby allowing an illicit migration of these proteins

into the internodal regions of the membrane; second, there

are high-affinity interactions that extend into the internodal

regions, and these are preserved in the CGT-null brain. We

speculate that interactions of NoGo receptor with its myelin-

associated ligands, including NoGo and MAG (Domeniconi

et al. 2002; Liu et al. 2002), are representative of this

adhesion.

Role of galactolipids in membrane structure and function

The absence of galactosylcerebroside and sulfatide in the

CGT-null mouse results in failure to form the transverse

bands normally associated with paranodal axoglial junctions

(Dupree et al. 1998). As a consequence, the axolemmal

proteins Caspr and Kv1 channels are delocalized (Fig. 7).

Here we have added, as a powerful diagnostic tool for the

fractionation of myelin and axolemmal proteins, a biochemi-

cal correlate to these data. In the CGT-null mouse there are

relatively high concentrations of axolemmal nodal, paranodal

and juxtaparanodal proteins in the main band and light

dispersion. These results suggest that the high-affinity

interactions between myelin and axolemma are intact, but

delocalized. Furthermore, these changes may be the result of

fundamental differences in the structure of the myelin

membrane compared with that in WT animals, possibly

involving the partitioning of proteins into glycosphingolipid

microdomains or ‘rafts’; perturbation of these microdomains

may have consequences for both the structure of myelin and

signaling between myelin and neurons (Taylor et al. 2002;

Marta et al. 2003). Evidence from remyelination, paranodal

mutants, and hypomyelinating or dysmyelinating mutant

animals has also suggested interactions between the axon and

the overlying myelin sheath (Rosenbluth 1987, 1990; Popko

2000; Uschkureit et al. 2000; Bhat et al. 2001; Mathis et al.

2001; Arroyo et al. 2002).

Key elements of the junctional complex may need to be

brought together in glycosphingolipid microdomains (‘lipid

rafts’) (Simons and Ikonen 1997; Taylor et al. 2003) in order

to form a normal, functional adhesive complex. This idea is

consistent with the fact that both Caspr and contactin are

detected at paranodes in CGT-null mice, albeit at much

lower densities than in WT animals, whereas NF-155 is

detected at much lower levels in CGT-null myelin and is not

detected at paranodes. This observation suggests that the

expression, trafficking and/or targeting of NF-155 depends

on galactolipids, and that loss of these membrane compo-

nents results in failure to establish the protein complexes

necessary for normal paranodal structure. Glycosphingoli-

pids are clearly important for signaling cascades in OLs and

other cells (Dyer and Benjamins 1990, 1991; Bansal et al.

1999; Taylor et al. 2003). For example, addition of anti-

GalC or anti-sulfatide antibodies to OL progenitors results in

the reversible inhibition of terminal differentiation (Bansal

et al. 1999); consistent with this, OL progenitors differen-

tiate more rapidly and to a greater extent in CGT-null

animals (Bansal et al. 1999; Marcus et al. 2000). There also

appears to be a metabolic relationship between MBP and

GalC because the Shiverer mutant mouse, which lacks MBP,

also has reduced levels of GalC (Bird et al. 1978; Uschkureit

et al. 2000). Finally, we have shown that several ‘myelin’

proteins are present at levels different from that seen in WT

animals (Kim and Pfeiffer 1999; Marcus et al. 2000);

specifically, Fyn and OSP are increased, whereas NF-155

is strongly decreased (Fig. 6). Thus, glycosphingolipids may

be key molecules in the structure and function of myelin and

neuroglial signaling.

In summary, we have shown that high-affinity protein–

protein interactions exist between myelin and axolemma, and

that these can be exploited to biochemically dissect myeli-

nated nerve fibers. Future experiments designed to identify

the protein complexes responsible for these interactions

should elucidate neuroglial signaling pathways important for

myelin formation, maintenance and function.

Acknowledgements

We are pleased to acknowledge the expert technical and adminis-

trative assistance of Ms. Susan Winkler, Janice Liseo, Jenifer

Gilman and Wendy Wolcott, and excellent discussions rendered by

Cecilia Marta and Michaela Anitei. We thank Dr James Trimmer

(SUNY Stony Brook, NY, USA) for antibodies against ion channels

and Dr Elior Peles (Weizmann Institute, Israel) for antibodies

against Caspr. This work was supported by grants from the National

Institutes of Health NS10861 (SEP), NS 41078 (SEP), NS38878

(RB), NS45440 (CT) and NS44916 (MNR). MNR is a Young

Investigator of the Wadsworth Foundation.



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The myelin–axolemmal complex: biochemical dissection and the role of galactosphingolipids

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