GLIA 00:000–000 (2013)
A Critical Role for the Cholesterol-Associated
Proteolipids PLP and M6B in Myelination of
the Central Nervous System
2
€
HAUKE B. WERNER,1* EVA-MARIA KRAMER-ALBERS,
NICOLA STRENZKE,3 GESINE SAHER,1
4
5
STEFAN TENZER, YOSHIKO OHNO-IWASHITA, PATRICIA DE MONASTERIO-SCHRADER,1
1
€
WIEBKE MOBIUS,
TOBIAS MOSER,3 IAN R. GRIFFITHS,6 AND KLAUS-ARMIN NAVE1*
1
Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Goettingen, Germany
2
Department of Molecular Cell Biology, Johannes Gutenberg University Mainz, Mainz, Germany
3
Inner Ear Lab, Department of Otolaryngology, Center for Molecular Physiology of the Brain,
University of Goettingen, Goettingen, Germany
4
Institute of Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
5
Faculty of Pharmacy, Iwaki Meisei University, Fukushima, Japan
6
Department of Veterinary Clinical Studies, University of Glasgow, Bearsden, Glasgow, United Kingdom
KEY WORDS
oligodendrocyte; myelin biogenesis; myelin proteome; cholesterol; Pelizaeus-Merzbacher disease; leukodystrophy;
spastic paraplegia
ABSTRACT
The formation of central nervous system myelin by oligodendrocytes requires sterol synthesis and is associated with a
significant enrichment of cholesterol in the myelin membrane. However, it is unknown how oligodendrocytes concentrate cholesterol above the level found in nonmyelin membranes. Here, we demonstrate a critical role for proteolipids
in cholesterol accumulation. Mice lacking the most abundant
myelin protein, proteolipid protein (PLP), are fully myelinated, but PLP-deficient myelin exhibits a reduced cholesterol
content. We therefore hypothesized that ‘‘high cholesterol’’ is
not essential in the myelin sheath itself but is required for an
earlier step of myelin biogenesis that is fully compensated for
in the absence of PLP. We also found that a PLP-homolog,
glycoprotein M6B, is a myelin component of low abundance.
By targeting the Gpm6b-gene and crossbreeding, we found
that single-mutant mice lacking either PLP or M6B are fully
myelinated, while double mutants remain severely hypomyelinated, with enhanced neurodegeneration and premature
death. As both PLP and M6B bind membrane cholesterol and
associate with the same cholesterol-rich oligodendroglial
membrane microdomains, we suggest a model in which proteolipids facilitate myelination by sequestering cholesterol.
While either proteolipid can maintain a threshold level of
cholesterol in the secretory pathway that allows myelin biogenesis, lack of both proteolipids results in a severe molecular imbalance of prospective myelin membrane. However,
M6B is not efficiently sorted into mature myelin, in which it
is 200-fold less abundant than PLP. Thus, only PLP contributes to the high cholesterol content of myelin by association
and co-transport. V 2013 Wiley Periodicals, Inc.
C
INTRODUCTION
Oligodendrocytes are highly specialized glial cells of
the vertebrate central nervous system (CNS), with cellular processes that spirally enwrap axons with a multiC 2013
V
Wiley Periodicals, Inc.
layered myelin sheath, which facilitates saltatory
impulse propagation and normal motor and sensory
functions (Nave, 2010). Myelin provides an attractive
model to study membrane lipids in vivo because it can
be biochemically purified, and its high content of lipids
(>70% of dry weight) and particularly of cholesterol
(>25% of total lipid) exceeds that of most other biological membranes (Chrast et al., 2011; Morell and Jurevics,
1996; Norton and Poduslo, 1973). The function of the
high lipid content of myelin is not known, but it is
widely assumed that it contributes to the property of
myelin as an electrical insulator (O’Brien, 1965). In
other cellular systems, cholesterol facilitates membrane
biogenesis, fluidity, and curving (Huttner and Zimmerberg, 2001) and assembles with glycosphingolipids in
lipid-rich membrane microdomains (rafts) (Simons and
Ikonen, 1997). As purified myelin has biochemical properties that fulfill raft criteria it has been suggested that
the generation, stabilization, and enlargement of
cholesterol-rich membrane microdomains in the secretory pathway is a key step in myelin biogenesis (Gielen
et al., 2006; Kramer et al., 2001).
We have previously shown that in mutant mice lacking cholesterol biosynthesis selectively in oligodendrocytes, CNS myelination depends on cholesterol uptake
from surrounding cells (Saher et al., 2005). This suggests that the availability of cholesterol is rate limiting
for myelination. However, it has remained unknown
how oligodendrocytes enrich membrane cholesterol to
the high level ultimately present in myelin, even though
the molecular mechanisms to establish and maintain
Grant sponsors: BMBF (DLR-Leukonet) and the European Commission (EUHealth FP7-LeukoTreat).
*Correspondence to: Hauke B. Werner, Department of Neurogenetics, Max
Planck Institute of Experimental Medicine, Hermann Rein Str. 3, D-37075 Goettingen, Germany. E-mail: [email protected] or Klaus-Armin Nave, Department of
Neurogenetics, Max Planck Institute of Experimental Medicine, Hermann Rein
Str. 3, D-37075 Goettingen, Germany. E-mail: [email protected]
Received 25 October 2012; Accepted 30 November 2012
DOI 10.1002/glia.22456
Published online in Wiley Online Library (wileyonlinelibrary. com).
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WERNER ET AL.
different cholesterol levels in various other cellular organelles have begun to be revealed (Maxfield and van
Meer, 2010).
The dominant protein of CNS myelin is a transmembrane tetraspan, proteolipid protein (PLP), which associates with cholesterol, at least in cultured oligodendrocytes (Simons et al., 2000). However, the deficiency of
PLP and its smaller splice isoform DM20 in mice does
not interfere with myelination (Klugmann et al., 1997).
We therefore hypothesized that PLP serves a role in
myelination that could be masked in PLP-deficient oligodendrocytes by other, functionally redundant tetraspan
proteins. We found that a PLP homolog, glycoprotein
M6B, is a myelin constituent of low abundance. M6B is
expressed in neurons and oligodendrocytes (Werner
et al., 2001; Yan et al., 1993), but its function is yet
unknown. We therefore targeted the Gpm6b gene in
mice and compared CNS myelination in mice that lack
either M6B, PLP, or both proteolipids. We find that the
expression of either PLP or M6B is required for CNS
myelination. Our data suggest that the common function
of both proteolipids in the biogenesis of myelin is related
to their capacity to associate with membrane cholesterol
in the oligodendroglial secretory pathway. Strikingly,
only PLP determines the high cholesterol content of
mature myelin by molecular association and co-transport because it is much more efficiently sorted into myelin and therefore >200-fold more abundant than M6B.
vial for peptide analysis via ultra-performance liquid
chromatography-mass spectrometry (UPLC-MS).
UPLC Configuration
Capillary LC of tryptic peptides was performed with a
Waters NanoAcquity UPLC system equipped with a
75 lm 3 150 mm BEH C18 RP column and a 2.6 lL
PEEKSIL sample loop (SGE, Darmstadt, Germany). The
aqueous mobile phase (mobile phase A) was H2O (LCMS Grade, Roth, Freiburg, Germany) with 0.1% formic
acid. The organic mobile phase (mobile phase B) was
0.1% formic acid in acetonitril (ACN) (LC-MS grade,
Roth). Samples (2.6 lL injection) were loaded onto the
column in direct injection mode with 3% mobile phase B
for 15 min at 400 nL/min, followed by an additional
10 min wash (3% B) at 300 nL/min. Peptides were eluted
from the column with a gradient from 3–35% mobile
phase B over 90 min at 300 nL/min followed by a 20 min
rinse of 80% mobile phase B. The column was immediately re-equilibrated at initial conditions (3% mobile
phase B) for 20 min. [Glu1]fibrinopeptide was used as
lockmass at 300 fmol/mL. Lockmass solution was delivered from the auxiliary pump of the NanoAcquity system
at 400 nL/min to the reference sprayer of the NanoLockSpray source. Samples were analyzed in quintuplicate.
Mass Spectrometer Configuration
MATERIALS AND METHODS
Myelin Purification
A light-weight membrane fraction enriched for myelin
was purified from mouse brains as described (Werner
et al., 2007). The protein concentration was determined
using the 2D-Quant kit (GE Healthcare). Mice for myelin protein and lipid analysis were 75 days old.
Protein Digest Preparation
For protein identification by nanoUPLC-MSE, myelin
fractions (20 lg total protein, n 5 4) purified from wild
type (WT) c57Bl6 mouse brains were precipitated using
the ProteoExtract Protein Precipitation Kit (Merck,
Darmstadt, Germany). Precipitated proteins were solubilized in 25 mM ammonium bicarbonate containing 0.1%
RapiGest (Waters, Eschborn, Germany; 80°C, 15 min),
followed by reduction with 5 mM dithiothreitol (DTT)
(45 min, 56°C) and alkylation of cysteines with iodoacetamide (Sigma, Taufkirchen, Germany; 15 mM, 25°C, 1 h
in dark). For digestions, 0.2 lg porcine sequencing grade
trypsin (Promega, Mannheim, Germany) was added, and
the samples were incubated overnight at 37°C. After
digestion, RapiGest was hydrolyzed by adding 10 mM
HCl (37°C, 10 min), and the resulting precipitate was
removed by centrifugation (13,000g, 15 min, 4°C), and
the supernatant was transferred into an autosampler
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MS analysis of tryptic peptides was performed using a
Waters Q-TOF Premier API system, operated in V-mode
with typical resolving power of at least 10,000 in positive ion mode as described (Kr€
amer-Albers et al., 2007;
Patzig et al., 2011).
Data Processing and Protein Identification
The continuum LC-MSE data were processed and
searched using the IDENTITYE-Algorithm of ProteinLynx Global Server (PLGS) version 2.3. Protein identifications were assigned by searching the Uni-ProtKB/
Swiss-Prot Protein Knowledgebase Release 56.0 for
mouse proteins (15.813 entries) supplemented with
known possible contaminants (porcine trypsin, human
keratins) using the precursor and fragmentation data
afforded by the LC-MSE acquisition method as described
(Kr€
amer-Albers et al., 2007; Patzig et al., 2011). Peptide
identifications were restricted to tryptic peptides with no
more than one missed cleavage. Carbamidomethyl cysteine was set as fixed modification, and oxidized methionine, protein N-acetylation, and deamidation of
asparagine and glutamine were searched as variable
modifications. Maximal mass deviations for database
search were set at 15 ppm for precursor ions and 30 ppm
for fragment ions. For a valid protein identification, the
following criteria had to be met: at least two peptides
detected with together at least seven fragments. The false
PROTEOLIPIDS SEQUESTER CHOLESTEROL IN MYELINATION
3
positive rate for protein identification was set to 3% based
on search of a randomized database, which was generated
automatically using PLGS 2.3 by randomizing the
sequence of each entry. By using technical replication rate
as a filter, the false positive rate was further minimized,
as false positive identifications do not tend to replicate
across injections due to their random nature. Quantification of the proteins was based on the three most intense
tryptic peptides as described (Patzig et al., 2011).
targeted allele was identified with P1 and reverse
primer P3 (50 -GCAATCCATCTTGTTCAATGGC), yielding a 700-bp fragment. PLPnull-mice were described previously (Klugmann et al., 1997). Mice were bred on the
c57Bl6/6J background for over 10 generations but formally are a c57Bl6/6J*129SV hybrid. Experiments were
in compliance with the animal policies of the MPI of Experimental Medicine, approved by the German Federal
State of Niedersachsen.
Molecular Cloning and Mouse Genetics
RNA Analysis
For gene targeting, we constructed a conventional
replacement vector in pSP72 (Promega). For the short
homologous arm, we PCR-amplified a 1.3-kb fragment at
the 30 -end of intron 3 that included the splice acceptor of
exon 4 from the cloned mouse (129SV) Gpm6b gene with
tailored primers to introduce Cla1 (50 ) and EcoR1 (30 )
restriction sites and an in-frame stop codon (primer
sequences available upon request). A 4.1-kb Nsi1 fragment containing parts of exon 5 and intron 5 became
the long homologous arm. For negative selection, a neomycin resistance cassette (neo) under control of the herpes simplex virus (HSV) thymidine kinase (tk) promoter
was used. Neo was subcloned with Xho1 and Sal1. For
positive selection, a Cla1 fragment of the HSV-tk under
control of the HSV-tk promoter was subcloned into the
vector. With this targeting strategy, we replaced most of
exons 4 and 5, including intron 4 by neo. As exon 4 of
the Gpm6b gene encodes the first membrane-spanning
domain of M6B and is the first exon present in all M6BmRNAs (Werner et al., 2001), the targeted Gpm6b allele
does not encode any membrane-spanning polypeptides.
R1 mouse embryonic stem cells (ES, provided by A.
Nagy) were electroporated with 50 lg linearized (Pvu1)
targeting vector in phosphate buffered saline (PBS)
(BioRad GenePulser, 240 V, 500 lF). Transfected ES (2
3 107) were cultured on gelatinized 10-cm dishes
(Falcon) for 1 day and then selected with 300 lg/mL
G418 and 2 lM Ganciclovir. On day 10 after electroporation, 192 resistant clones were picked and three with homologous recombination were identified by polymerase
chain reaction (PCR). ES were microinjected into
c57Bl6/6J blastocysts (3.5 dpc), and embryos were transferred to pseudo-pregnant foster mothers. Twelve highly
chimeric males were obtained and bred with c57Bl6/6J
females. We interbred heterozygous offspring to obtain
homozygous mutants, which were born at the expected
frequency. M6Bnull-mice are viable and fertile. Genomic
DNA was isolated from tail biopsies using the DNeasy96
kit (Qiagen). Southern blot was performed using standard procedures. To exclude additional illegitimate recombination, genomic DNA after EcoR1, BamH1, or Xba1
restriction was hybridized with a radioactively labeled
neo-probe. For routine genotyping by PCR co-amplification, presence of the Gpm6b WT-allele was shown using
forward primer P1 (50 -CCCTTTGCCT CCCAGTCAGTTG) and reverse primer P2 (50 -CCAGGGAGGC
ATAGGGAACT), which yielded a 400-bp fragment. The
Total brain RNA (n 5 2 per genotype) was extracted
using Qiazol (Qiagen). cDNA was synthesized using random nonamer primers and Superscript III RNase H
reverse transcriptase (Invitrogen). Equal cDNA concentration was verified by RT-PCR for cyclophilin (forward
50 -ACCCCACCGTGTTCTTCGA and reverse 50 -CATTTG
CCATGGACAAGATG). RT-PCR primers were specific for
Gpm6b exon 1A (forward 50 -CTCCCGCCAGTCTCCAAC
CATGG) or exon 1B (forward 50 -TATGGTCGCCTGCTC
CTTG) and exon 4 (reverse 50 -CCAGGGAGGCATAGGG
AACT) or neo (reverse 50 -GCAATCCATCTTGTTCAAT
GGC). All reactions were carried out in duplicate. Fragment identity and in-frame translation termination were
proven by molecular sequencing.
Motor Function Test
Mice (n 5 6 per genotype) were tested for motor capabilities on a rotating rod (diameter 3 cm). All mice were
placed on the roller at rest. After 15 s, the rotarod was
set at 1 round per min (rpm). In 60 s intervals, the
rotating speed was successively increased to 2, 4, 8, 12,
16, and 20 rpm. In a series of five trials per animal, the
latency to fall off was measured.
Antiserum Generation
An antiserum specific for the C-terminal x-domain of
M6B was previously described (Werner et al., 2001). A
novel antipeptide serum specific for the N-terminal a-domain of M6B was newly generated (Eurogentec) using
the epitope KPAMETAAEEN.
Membrane Microdomain Extraction
Membrane microdomain extraction was performed as
described (Kr€
amer-Albers et al., 2006; Simons et al.,
2000). Briefly, myelin was resuspended in extraction
buffer (50 mM Tris/HCl, pH 7.4, 5 mM ethylenediaminetetraacetic acid (EDTA), 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) supplemented
with protease inhibitors, or cultivated oligodendrocytes
were washed in Tris-buffered saline (TBS), scraped into
300 ll of extraction buffer and extracted for 30 min at
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4
WERNER ET AL.
4°C. The extract was adjusted to 30% Optiprep (600 ll)
(Sigma, Deidenhofen, Germany), overlaid with 20% Optiprep in extraction buffer (1.4 mL) and with extraction
buffer only (200 ll), followed by ultracentrifugation for 2 h
at 55,000 rpm in a TLS-55 rotor (Beckman, Fullerton,
CA). Six fractions of 360 ll were collected from the top.
Immunoblotting
Immunoblotting was performed as described (Werner
et al., 2007). Antibodies were specific for M6B (a-domain,
1:200), PLP/DM20 (A431; 1:5,000; Jung et al., 1996),
SIRT2 (Santa Cruz, 1:2,000), cyclic nucleotide phosphodiesterase (CNP; Sigma, 1:1,000), myelin oligodendrocyte
glycoprotein (MOG, 8-18C5; (kindly provided by Chris
Linington, 1:5,000), caveolin-1 (Pharmingen, 1:50), plasmolipin (kindly provided by F. Bosse) (Bosse et al., 2003)
(1:500), myelin and lymphocyte protein (MAL; TM-1,
1:100; kindly provided by N. Schaeren-Wiemers)
(Schaeren-Wiemers et al., 2004), septin 6 (SEPT6; Buser
et al., 2009), oligodendrocyte specific protein (OSP)/claudin-11 (Zymed, 1:500), CD81 (Pharmingen), CD9 (Pharmingen, 1:500), myelin-associated glycoprotein (MAG)
(Chemicon, 1:50), Flotillin (Pharmingen, 1:250), N-cadherin (Pharmingen, 1:2,500), or a-tubulin (Sigma, 1:2,000).
1:750), GFAP (Chemicon 1:500), CM1 (Santa Cruz,
1:500), MIB5 (Dianova, 1:100), MAG (Chemicon, 1:50),
gm130 (Pharmingen, 1:100). Gallyas silver impregnation
of myelin was performed as described (Lappe-Siefke
et al., 2003). Sections were analyzed by bright-field light
microscopy (Zeiss Axiophot) at 53 and 403 magnification. Images were captured using a Hamamatsu CCD
camera, KAPPA ImageBase control 2.7.2, and ImageJ
1.410, and processed in Photoshop TIF format.
Electron Microscopy
Cryo-immuno electron microscopy (EM) was performed
as described (Werner et al., 2007). Antibodies were specific for PLP (A431; 1:250) (Jung et al., 1996), M6B (adomain, 1:100), or g-adaptin (BD Biosciences 610385,
1:65). For transmission EM, we perfused mice with 2%
glutaraldehyde and 4% paraformaldehyde (PFA) in PBS.
Tissues were postfixed in 1% OsO4 in 0.1 M sucrose and
embedded in epoxy resin (Serva). Ultrathin sections were
contrasted with uranylacetate before examination in a
LEO EM 912AB electron microscope (Zeiss, Oberkochen,
Germany). Pictures were taken with an on-axis 2048 3
2048-CCD-camera (Proscan, Scheuring, Germany).
Cell Culture and Transfection
Lipid Analysis
Thin layer chromatography of myelin (n 5 2 animals
per genotype) was as described (Lappe-Siefke et al., 2003).
Quantification of myelin sterols (n 5 3 animals per genotype) was as described (Keller et al., 2004). Briefly, lipids
were extracted with choloroform/methanol (1:1, v/v). Lipids were saponified with 20% (w/v) potassium hydroxide
in 66% aqueous methanol solution for 1 h at 100°C in the
presence of an internal standard (dehydrocholesterol).
Hydrolyzed samples were extracted twice with equal volumes of petroleum ether, and the pooled extracts were
backwashed with 5% (v/v) aqueous acetic acid and then
taken to dryness. Nonsaponifiable lipids were dissolved in
mobile phase solvent (methanol–isopropanol, 7:1, v/v), and
aliquots were subjected to reverse-phase high-performance
liquid chromatography (HPLC) (Shandon Hypersil ODS
column, 2.1 mm 3 200 mm, flow rate 0.2 mL/min) with
online ultraviolet (UV) detection at either 214 or 282 nm
(Smart system, Amersham Pharmacia). A standard curve
using cholesterol was used to quantify sterol mass in cell
extracts after normalization to the internal standard.
Histology
Immunohistochemistry (IHC) analysis (n 5 6 mice per
genotype and age, at multiple sectioning levels) was as
described (Werner et al., 2007). Antibodies were specific
for M6B (a-domain, 1:500), PLP (A431, 1:600) (Jung
et al., 1996), CNP (Sigma, 1:300), MAC3 (Pharmingen,
1:100), amyloid precursor protein (APP) (Chemicon,
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The cell line Oli-neu (Jung et al., 1994) was cultured
on poly L-lysine-coated coverslips in Sato medium containing 5% horse serum. Transfections were performed
with FuGENE6 (Roche) with 1 lg of plasmid DNA in
1 mL medium. The expression vectors PLP-enhanced
green fluorescent protein (EGFP), PLP-enhanced yellow
fluorescent protein (EYFP), and Neuroligin-EGFP were
described previously (Dhaunchak and Nave, 2007;
Werner et al., 2007). The expression vector M6B-EGFP
was generated by amplifying M6B cDNA (splice isoform
abTMDx) with tailored primers omitting the translation
termination codon and introducing restriction sites for
cloning into pEGFP-N1 (Clontech). M6B-EGFP localization and trafficking did not differ from untagged M6B
(data not shown). For the process outgrowth assay, cells
were analyzed 30 h after transfection. For perfringolysin-O-labeling of cholesterol-rich membrane microdomains (Waheed et al., 2001), cells were analyzed 36 h after transfection. Cells were washed on ice, blocked for
10 min with 1% bovine serum albumin (BSA), and incubated for 30 min with biotinylated perfringolysin-O in
PBS (0.015 mg/mL). Then, cells were fixed with 4% PFA
and incubated with anti-biotin antibodies (1:100, Dianova) for 2 h at room temperature (RT) and Cy3 goat
anti-IgG mouse antibodies (1:2,000, Dianova) for 1 h at
RT. After washing with PBS, cells were mounted with
Aqua-Poly/Mount (Polysciences, Warrington, PA) on
glass slides. Confocal images were captured using a
Leica TCS-SP2 microscope (Leica Microsystems, Heidelberg, Germany) with a 633 oil objective and Leica confocal software (LCS Lite 2.61). For image analysis, ImageJ
5
PROTEOLIPIDS SEQUESTER CHOLESTEROL IN MYELINATION
1.383 (W. Rasband, NIH, USA) was used with the plugins Spectral Unmixing for ImageJ v1.2 (J. Walter), Cell
Counter (K. De Vos), and NeuronJ 1.3.1 (E. Meijering).
Photocholesterol Labeling
The synthesis of [3a-3H]-6,6-azocholestan-3b-ol (referred
to as photo-cholesterol) has been described (Kr€
amer-Albers
et al., 2006; Thiele et al., 2000). For preparation of
the [3H]-photo-cholesterol/methyl-b-cyclodextrin (MbCD)
inclusion complex the steroid (final concentration 0.3 mM)
was added to an aqueous solution of MbCD (40 mg/mL).
The mixture was overlaid with N2 and continuously
vortexed under light protection at 30°C for 24 h in a thermomixer. Labeling of cultured oligodendrocytes was performed as described (Kr€
amer-Albers et al., 2006). Cells
were washed with Sato medium supplemented with 1%
horse serum (HS), which was depleted of lipids (dlHS), and
incubated with 100 ll (50lCi) [3H]-photo-cholesterol–
MbCD inclusion complex in 2 mL Sato/1% dlHS for 16 h.
Cells were irradiated for 20 min with UV light at 4°C and
then disrupted in lysis buffer (25 mM Tris/HCl pH 7.4, 150
mM NaCl, 1% Triton X-100) supplemented with protease
inhibitors for 15 min on ice, followed by a brief spin (10
min, 200g) to deplete nuclei. A small sample of the lysate
was directly processed for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography,
while the rest was subjected to immunoprecipitation with
antibodies specific for PLP (A431) (Jung et al., 1996) or
M6B (x-domain) (Werner et al., 2001).
Auditory Brain Stem Response and
Visual Evoked Potentials
Young adult mice aged 9–11 weeks or old mice aged
16–25 weeks were anesthetized by i.p. injections of avertine (14 g/l Tribromethanol (Sigma-Aldrich) in 1.13%
tert-Amylethanol (Merck) in distilled water, used at 20
mL/kg). For auditory brainstem response (ABR), tone
bursts (4/6/8/12/16/24/32 kHz, 10-ms plateau, 1-ms rise/
fall) or clicks (0.03 ms) were applied in the free field at
20 Hz (System 2, Tucker-Davis Technology, Gainesville,
FL) using a high-frequency speaker (Monacor International, Bremen, Germany). Intensities are displayed as
sound pressure level (dB root mean square for tone
bursts, dB peak equivalent for clicks). The difference
potential between vertex and mastoid subdermal needles
was amplified 50,000 times, filtered (low pass: 4 kHz,
high pass: 400 Hz), and sampled at a rate of 50 kHz for
20 ms, 2,000 times twice to obtain two mean ABR traces
for each sound intensity. Thresholds were estimated
with 10-dB precision by visual inspection by two trained
investigators. For distortion product otoacoustic emissions (DPOAE), a CD player was used to generate two
primary tones (f1 and f2) with a ratio of f2/f1 of 1.2 and
f2 ranging from 6 to 20 kHz. Primary tones were
coupled into the ear canal by a custom-made probe
containing a MKE-2 microphone (Sennheiser, Hannover,
Germany) and two ED1/EC1 speakers (Tucker-Davis)
and adjusted to an intensity of 60 dB at the ear drum.
The microphone signal was amplified (DMP3, MIDIMAN), sampled by a 24-bit sound card (TerraTec, Nettetal, Germany) and Sound Forge software (Sony), and
analyzed by fast Fourier transformation using Matlab
software. Animal numbers were 3 for PLPnull*M6Bnull, 2
for PLPnull, and 5 for M6Bnull for ABR in young animals,
5 for PLPnull*M6Bnull, 8 for PLPnull, and 8 for M6Bnull
for ABR in older adult animals, 6 for PLPnull*M6Bnull, 6
for PLPnull, 5 for M6Bnull for hearing threshold in young
animals, and 5 for PLPnull*M6Bnull, 6 for PLPnull, 7 for
M6Bnull in older adult animals. For visual evoked potentials (VEP), the right eyes of anesthetized mice were
exposed to 2-ms light flashes emitted from a light diode
(Nichia White LED, model NSPW500BS, Tokushima, Japan) at a rate of 2 Hz. The difference potential between
the frontal midline and the left occipital area was amplified 50,000 times, low-pass filtered (1,000 Hz), and
sampled at a rate of 10 kHz for 100 ms, and averaged at
least 2 3 200 times for each animal. Animal numbers for
VEP were 6 M6Bnull, 8 PLPnull, and 5 PLPnull*M6Bnull.
RESULTS
Subcellular Localization of the Oligodendroglial
Proteolipids PLP and M6B
We followed the hypothesis that mice lacking the most
abundant protein of CNS myelin (PLP) display a fairly
mild neurological phenotype because its homolog M6B
may functionally overlap with PLP in myelin biogenesis.
To determine the subcellular localization of PLP and
M6B in oligodendrocytes and myelin, we performed
cryo-immuno EM. As expected, PLP-immunolabeling
was abundant in compact myelin (Fig. 1A) where M6Blabeling was also detected, although at lower abundance
(Fig. 1B). M6B-labeling was more abundant in early oligodendroglial membrane sheaths engulfing axons prior
to compaction (not shown) suggesting diminished incorporation of M6B upon myelin maturation. Within oligodendroglial cell bodies, most PLP and M6B labeling was
associated with clathrin-coated, Golgi-derived transport
vesicles that colocalize with g-adaptin (Fig. 1C,D). Thus,
the distribution of PLP and M6B overlaps in myelin and
in the oligodendroglial secretory pathway.
Relative Abundance of Tetraspan Proteins
in CNS Myelin
Cryo-immuno EM does not allow to reliably determine
relative protein abundances because of differences in the
accessibility of antibodies to different cellular compartments and antibody–antigen affinity. To determine the relative abundance of PLP and M6B, we subjected purified
CNS myelin to separation by nanoscale one-dimensional
UPLC (1D-UPLC), protein identification by quadrupole
time-of-flight mass-spectrometry, and data acquisition
using an alternating low and elevated collision energy acquisition mode (LC-MSE). LC-MSE yields information on
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6
WERNER ET AL.
Fig. 1. Proteolipids PLP and M6B are associated with CNS myelin
and a late/post Golgi compartment in oligodendrocytes. Immunodetection of PLP (A, C) and M6B (B, D) visualized with 10 nm gold particles
in myelin (A, B) and oligodendrocyte somata (C, D) on spinal cord sections. Note that both PLP and M6B (black arrowheads) are associated
with mature compact myelin, although the labeling intensity differs.
Within oligodendrocytes, PLP and M6B were mostly confined to a late/
post Golgi compartment that co-labeled with g-adaptin (white arrowheads pointing at 15 nm gold particles in insets in C0 ,D0 ).
the relative abundance of proteins (Jahn et al., 2009). PLP
and M6B constituted 15.43% and 0.05% of the total myelin
protein, respectively (Table 1). Several other tetraspans
were also detected, namely claudin 11 (0.7%), CD81
(0.26%), Plasmolipin (0.15%), TSPAN2 (0.08%), and CD82
(0.06%). The latter tetraspans are unrelated to PLP by
amino acid sequence, while M6B shares 57% sequence
identity (72% similarity) with PLP. Thus, despite its low
relative abundance in mature myelin, M6B was a plausible candidate to test for a functional overlap with PLP.
PLP and M6B Share Common Biochemical
Features
GLIA
The association of membrane proteins with cholesterol
occurs via sequence consensus motifs such as the cholesterol recognition/interaction consensus (CRAC) and the
sterol sensing domain (SSD) (Epand, 2006). In PLP, a
CRAC is located at the cytoplasmic interface of TMD2
(residues 86–98-L-AEGF-Y-TTGAV-R-), and a central
sequence motif of the SSD is present at the extracellular
7
PROTEOLIPIDS SEQUESTER CHOLESTEROL IN MYELINATION
TABLE 1. Relative Abundance of Tetraspan-Transmembrane Proteins in Myelin
Protein name
Accession
Myelin tetraspans
Myelin proteolipid protein
P60202
Claudin 11 (OSP)
Q60771
CD81
P35762
Plasmolipin (TM4SF11)
Q9DCU2
Tetraspanin 2
Q922J6
CD82 (KAI1)
P40237
Glycoprotein M6B
P35803
Myelin and lymphocyte protein
O08819
CD9
P40240
Selected nontetraspan proteins in myelin
Myelin basic protein
P04370
CNP
P16330
MOG
Q61885
MAG
P20917
Sirtuin 2
Q8VDQ8
Carbonic anhydrase 2
P00920
MOBP
Q9D2P8
CD47
Q61735
Cell division control protein 42
P60766
SNAP23
O09044
Vesicle ass. membrane protein 2
P63044
Vesicle ass. membrane protein 3
P63024
Gene
%
RSD
TMHMM
Phobius
Reference
Plp1
Cldn11
Cd81
Pllp
Tspan2
Cd82
Gpm6b
Mal
Cd9
15.43
0.70
0.26
0.15
0.08
0.06
0.05
ND
ND
2.10
0.15
0.07
0.02
0.02
0.00
0.02
ND
ND
4
4
4
4
4
4
4
4
4
4
3
4
4
4
4
4
4
4
Klugmann et al., 1997
Gow et al., 1999
Terada et al., 2002
Bosse et al., 2003
Birling et al., 1999
Mela and Goldman, 2009
This paper
Schaeren-Wiemers et al., 2004
Kagawa et al., 1997
Mbp
Cnp1
Mog
Mag
Sirt2
Car2
Mobp
Cd47
Cdc42
Snap23
Vamp2
Vamp3
6.80
3.68
1.22
0.88
0.76
0.12
0.10
0.05
0.03
0.02
0.01
0.01
0.81
0.55
0.18
0.06
0.06
0.03
0.02
0.02
0.01
NA
NA
NA
0
0
3
1
0
0
0
5
0
0
1
1
0
0
2
1
0
0
0
5
0
0
1
1
Nawaz et al., 2009
Edgar et al., 2009
Johns and Bernard, 1999
Quarles, 2007
Werner et al., 2007
Cammer et al., 1976
Montague et al., 2006
Gitik et al., 2011
Thurnherr et al., 2006
Feldmann et al., 2009
Feldmann et al., 2009
Feldmann et al., 2009
Proteins identified by LC-MSE in myelin purified from mouse brains were subjected to systematic transmembrane domain prediction. All tetraspan-transmembrane proteins are given with their relative abundance in percent (%), the relative standard deviation (RSD), and the number of transmembrane domains predicted using the algorithms TMHMM and Phobius. Note that MAL and CD9 were not detected (ND) due to atypically distributed cleavage sites. Selected known non-tetraspan myelin proteins
are given for comparison. One selected reference each is given.
interface of TMD3 (residues 175–178-YIYF-) (Fig. 2A).
In M6B, a CRAC is located at the cytoplasmic interface
of TMD2 (residues 86–98-L-AEGF-Y-TTSAV-K-; Fig. 2B).
To test whether both PLP and M6B indeed bind cholesterol, cultured primary oligodendrocytes were labeled
with a 3H-labeled cholesterol derivative. This ‘‘photocholesterol’’ (but not photo-phosphatidyl-choline) can be specifically crosslinked to closely spaced cholesterol-associated proteins by UV light (Thiele et al., 2000). After photoactivation, membrane proteins were solubilized and those
crosslinked to photocholesterol were detected by autoradiography. In oligodendrocyte lysates, proteins with the mobility of PLP or glycosylated M6B were labeled (Fig. 2C).
Similar to PLP ((Simons et al., 2000) and Fig. 2C), we
could immunoprecipitate M6B*cholesterol adducts from
such lysates using antibodies specific for M6B (x-domain;
compare Fig. 2B). Thus, both PLP and M6B are cholesterol-associated proteins in oligodendrocytes.
This interaction could also be visualized in the oligodendrocyte progenitor cell line Oli-neu (Jung et al.,
1994). Oli-neu cells were transfected to overexpress PLP
or M6B tagged with a C-terminal EGFP moiety. By confocal imaging, we found many PLP-EGFP and M6BEGFP positive puncta costained with perfringolysin-O
(Fig. 2D,E). This nonlytic derivative of a Clostridium
perfringens toxin specifically labels cholesterol-rich
membrane domains (Waheed et al., 2001). Together, this
indicates that either proteolipid can mediate the enrichment of membrane-cholesterol. The functionality of the
motifs has not been tested by mutational analysis
because virtually all PLP mutations affecting TMDs
cause protein misfolding and retention in the endoplasmic reticulum (Dhaunchak and Nave, 2007). In particular, mutations interfering with the YIYF motif in the
SSD of human PLP cause the severe connatal form of
Pelizaeus-Merzbacher disease (Mimault et al., 1999).
To determine whether PLP and M6B are associated
with the same membrane microdomains, cultured
mouse oligodendrocytes and myelin were solubilized
and fractionated by density gradient centrifugation.
Similar to PLP (Simons et al., 2000), M6B was confined to a CHAPS-insoluble, Triton X-100-soluble membrane fraction (Fig. 2F,G). Thus, both proteolipids reside in a similar lipid milieu in oligodendrocytes and
in myelin.
High-Level Proteolipid Expression Induces
Process Formation
High-level expression of PLP and M6B (see above)
had unexpected consequences for the morphology of Olineu cells. Cells transfected with plasmids encoding PLP
or M6B developed longer and more branched processes
(Fig. 3A) than control cells transfected with a plasmid
encoding the unrelated transmembrane protein
Neuroligin-1. The cellular processes exhibited cholesterol-rich membrane domains (see above) and were covered with numerous outward-projecting membrane
tubules (Fig. 3A,B). Together, this indicates that highlevel expression of proteolipids enhances oligodendroglial membrane growth and process formation.
Myelination in Mice Lacking Proteolipid M6B
To investigate the function of M6B in vivo, we inactivated the X-linked Gpm6b gene by homologous
recombination in ES cells (Fig. 4A). After germ-line
transmission of the M6Bnull-allele, hemizygous male
and homozygous female mice were born at the
expected frequencies. Gene targeting was verified by
GLIA
Fig. 2. PLP and M6B bind cholesterol and associate with the same
membrane microdomains. (A, B): Proposed topological model of the oligodendroglial proteolipids PLP (A) and M6B (B). Proteolipids are hydrophobic proteins with four transmembrane domains (TMD) and cytoplasmic
N-and C-terminal domains. Amino acids conserved between PLP and
M6B are represented by black dots. Divergent amino acids (white dots)
are more frequent outside than within TMDs. Domains unique to either
proteolipid arise from alternative splicing (gray dots). M6B does not have
a domain with homology to the PLP-specific domain, and PLP/DM20 do
not have domains with homology to the a- and x-domains of M6B. Two disulfide bonds (
SS
) are conserved in the second extracellular loop. A
putative cholesterol recognition/interaction amino acid consensus motif
(CRAC) is conserved between PLP and M6B at the cytoplasmic interface
of TMD2. A central sequence motif (YIYF) of a putative SSD is present in
PLP at the extracellular interface of TMD3. (C): Primary oligodendrocytes were cultivated with 3H-photocholesterol and crosslinked by UV
irradiation. Labeled proteins in the total cell lysate were visualized by
fluography. After immunoprecipitation of the cell lysates with antisera
specific for PLP/DM20 (A431, left panel) or M6B (x-domain, right panel),
proteolipids were detected by fluography. (D, E): Proteolipids coassemble
with cholesterol-rich membrane domains in processes of oligodendroglial
cells. PLP-EGFP (D) or M6B-EGFP (E) were exogenously expressed in
Oli-neu cells and confocally imaged. In the cellular processes, colabeling
was observed with Perfringolysin-O, a marker for cholesterol-rich membrane domains. Oli-neu cells were stained live so that only Perfringolysin-O at the cell surface was visualized. (F, G): M6B and PLP are associated with a CHAPS-insoluble membrane fraction. A myelin-enriched
brain fraction (F) or cultivated oligodendrocytes (G) were solubilized with
CHAPS or Triton X-100 and floated in a density gradient. Fractions were
obtained from the top to the bottom (1–6), separated by SDS-PAGE, and
blotted. M6B was detected in the CHAPS-insoluble (lane 1) and Triton-insoluble (lane 4–6) fractions. PLP/DM20 was detected in the same fractions although the signal was much stronger when the same amount of
material was loaded. As a control, N-CAM120 was recovered from a Triton-insoluble fraction. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
9
PROTEOLIPIDS SEQUESTER CHOLESTEROL IN MYELINATION
Fig. 3. Proteolipid expression induces oligodendroglial process outgrowth. (A): PLP-EGFP, M6B-EGFP, or the unrelated control transmembrane protein Neuroligin-EGFP (ctrl) were exogenously expressed
in Oli-neu cells and imaged by confocal microscopy. (B): Total process
length, number of main processes (1st, 2nd, and 3rd branch), and outward-projecting membrane tubules were quantified (three independent
experiments, with minimum 50 cells quantified per construct). Data
represent the mean (6 SEM), Mann–Whitney U test of significance
was performed (****, P<0.0001). Note that expression of either proteolipid but not Neuroligin induced process formation in Oli-neu cells.
[Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Southern blotting (Fig. 4B) and genomic PCR (Fig.
4C), and the loss of M6B-expression was confirmed by
RT-PCR (Fig. 4D), IHC (Fig. 4E), and immunoblotting
(Fig. 4F). M6Bnull-mice showed no obvious behavioral
abnormality, were fertile, and had a normal life span.
We tested motor functions at 3, 12, 16, and 24 months
using a rotarod but detected no difference between
mutants and WT littermate controls (not shown).
Myelination of the optic nerve, corpus callosum, and
spinal cord was analyzed by EM. M6Bnull-mice were
fully myelinated, and myelin sheaths appeared of normal thickness (Fig. 4G,H). However, we observed that
the adaxonal glial membrane appeared occasionally
‘‘fused’’ with the axonal membrane (Fig. 4H), suggesting
condensation of the periaxonal space (not quantified).
Interestingly, a similar loss of extracellular membrane
spacing is a feature of compact myelin in PLPnull-mice,
in which the (double) intraperiod lines appear fused
(Klugmann et al., 1997; Mobius et al., 2008). We also
observed occasional double-myelinated axons and invaginations of oligodendroglial plasma membrane into the
axon (Fig. 4I). Taken together, M6B appears largely dispensable for overall neural development, including CNS
myelination. However, the occurrence of subtle ultrastructural defects in M6Bnull-mice suggested a function
in myelination that would be more obvious in doublemutant mice.
Lethal Developmental Defects of Mice Lacking
PLP and M6B
The obvious candidate for a genetic compensation of
M6B-function is PLP, the loss of which (in PLPnull-mice)
is also remarkably well tolerated in brain development
(Klugmann et al., 1997). To test this hypothesis, we
crossbred M6Bnull and PLPnull-mice. Meiotic recombination between these two X-linked genes (distance 29.4
Mb) was achieved and subsequent breeding yielded double mutant males (PLPnull*M6Bnull, also referred to as
double knockout, dKO).
PLPnull*M6Bnull-mice developed to term normally but
exhibited a severe neurological disease of early onset. 10
days old mice showed hindlimb spasticity, and mice at
all ages tested were unable to stay on a horizontal bar,
unless physically supported (Fig. 5A,B). They failed to
grasp objects with their forelimbs, and ataxia and
tremor were present. At 2–3 months, PLPnull*M6Bnullmice always had spastic hindlimbs, best seen on footprints (Fig. 5C), and developed a hunchback. Motor
defects were obvious when quantified with a rotarod at
2.5 and 4 months (Fig. 5D). Most PLPnull*M6Bnull mice
died prematurely at 4–5 months, and none could be
maintained for longer than 6 months even when food
and water were supplied at the cage floor (Fig. 5E). A
progressive disease was also observed in mosaic females
GLIA
Fig. 4. Targeted inactivation of the Gpm6b gene. (A): Exon 4 of the
WT Gpm6b gene encodes TMD1 and is the first exon present in all
M6B-transcripts. The targeting vector introduces a neomycin cassette
(neo) in sense orientation, replacing most of exons 4 and 5, including
intron 4. The targeted allele is depicted below. Mutant transcripts bear
a translation stop codon in the M6B-reading frame, abolishing coding
capacity for any TMD. Restriction sites for molecular cloning and genotyping primer positions are indicated. (B): Southern blot analysis of a
WT control male (1/Y), a heterozygous female (1/2), and a hemizygous
M6Bnull male (2/Y). A probe specific for the neomycin resistance cassette hybridized with the predicted 5.4 kb genomic fragment, indicating
that no illegitime integration occurred. (C): PCR analysis of genomic
DNA from a representative litter including heterozygous (1/2) and WT
(1/1) females, hemizygous (2/Y) M6Bnull males and WT (1/Y) controls.
The sense primer (P1) was specific for M6B intron 3, antisense primers
were specific for M6B exon 4 (P2) or neomycin (P3). P1 and P2 amplify
a WT fragment of 400 bp, P1 and P3 amplify a fragment of 700 bp indicating the targeted allele. (D): RT-PCR of total brain cDNA with primers specific for exons 1A and 4 or exons 1B and 4. M6B transcripts
could not be amplified from M6Bnull but from control animals. Multiple
fragments represent alternative splicing. (E): Sections of 2.5-monthsold WT and M6Bnull brains were subjected to IHC with an antiserum
directed against the N-terminal a-domain of M6B. M6B labeling (in
brown) was strong in the white matter (exemplified in the corpus callosum, top left). Outside the white matter, the ventricular surface was
labeled (lower images). Note absence of M6B labeling from M6Bnull.
Nuclei were counterstained (blue). (F): Myelin-enriched fractions from
brain homogenates of M6Bnull, PLPnull, and PLPnull*M6Bnull (dKO)
mice (age 2.5 months) were separated by SDS-PAGE and blotted. The
antiserum against M6B (a-domain) and a PLP/DM20-specific antiserum
were used for detection. MOG was detected as loading control. (G):
Electron microscopy of M6Bnull CNS at different ages revealed the presence of normal amounts of myelin. The representative example is from
the spinal cord of a 2.5-month-old animal. (H, I): Higher magnification
revealed irregularities at the axon–myelin interface, including irregular
width of the extracellular periaxonal space (P) (in H). Both regular
width and condensation (arrowheads) were often observed at the same
axon, suggesting loss of a strut-like function. The ultrastructure of compact myelin (M) appeared unaltered. Occasionally, the axon–myelin
apposition was severely disturbed (I). The example features double
myelination (**) of a unit of two axons (A) individually ensheathed by
compact and noncompact myelin (*). Invaginations (Inv) of membrane
into the axoplasm were a rare feature in M6Bnull, likely secondary to
myelin–axon apposition instability. These features were not quantified.
I, inner loop (adaxonal myelin layer). [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
PROTEOLIPIDS SEQUESTER CHOLESTEROL IN MYELINATION
11
Fig. 5. Impairment and early death of mice lacking PLP and M6B.
(A): As for WT or PLPnull controls, M6Bnull mice (age 2.5 months) show
normal motor capabilities when placed on a rod. (B): PLPnull*M6Bnull
(dKO) display progressive spasticity of the hindlimbs, which is more
moderate for the forelimbs. By holding them at the tail, they can be
placed on a rod but fall off as soon as the tail is released. The spastic
paw is illustrated in the inset. (C): Mouse hindpaws (2.5 months) were
inked and mice were placed on white paper. Paw-prints show that toes
of dKO are spastically spread when compared with WT or single-mutant controls. (D): Motor capabilities of dKO (green) were compared
with PLPnull (blue) and M6Bnull (red) littermates at 2.5 and 4 months
of age in the rotarod test. Double mutants cannot balance on the rod
even when it is not rotating, whereas single mutants improve motor
abilities with training. (E): Mortality rate in a Kaplan-Meyer plot (n 5
25 per genotype). While PLPnull and M6Bnull have a normal life span,
dKO died at 6 months of age at the latest. (F, G): Auditory function in
proteolipid mutants. (F): Grand averages of auditory brain stem
responses (ABR) (6 SEM) to 80 dB click stimuli from young adult mice
at 9–11 weeks. Wave I represents transmission in the peripheral part
of the auditory pathway and waves II–IV represent CNS transmission.
Note the wave latency shift, suggesting delayed signal transmission in
dKO, was statistically significant as determined by unpaired two-sided
t-test (*, P<0.05). (G): Averaged hearing thresholds in response to tone
bursts of different frequencies or click stimuli as determined by ABR
recordings. Results are given as decibel sound pressure level (dB SPL).
Statistical significance was determined by unpaired two-sided t-test (*,
P<0.05; **, P<0.01). Note that the hearing threshold in dKO is elevated compared with PLPnull and M6Bnull. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
(PLP2/2*M6B1/2 and PLP1/2*M6B2/2), but was milder
at 4–6 weeks than in PLPnull*M6Bnull-males, and led to
premature death at 7–12 months.
up to the cochlear ganglion, which is part of the peripheral nervous system (PNS) and myelinated by Schwann
cells. In contrast, waves II–V (CNS, myelinated by oligodendrocytes) were significantly delayed in PLPnull*
M6Bnull-mice at 2.5 months (Fig. 5F). The delay was
less pronounced at 4.5 months (not shown), likely
reflecting other age-dependent changes of hearing in all
genotypes. At both ages tested, wave III was most
delayed. At 4.5 months, waves II-V appeared delayed
also in PLPnull, but this did not reach significance.
Functional Defects of Myelinated Fiber Tracts
To evaluate signal propagation, we determined ABR to
tone bursts and click stimuli. The latency of wave I was
equal in all groups, reflecting normal auditory activity
GLIA
12
WERNER ET AL.
ABR-latencies and amplitudes obtained from M6Bnullmice were equal to those previously measured for WTs
with a similar genetic background (Eiberger et al., 2006;
Sons et al., 2006).
Evaluation of hearing thresholds revealed a moderate
pantonal hearing loss across all frequencies in
PLPnull*M6Bnull-mice (Fig. 5G and data not shown).
PLPnull-mice at 4.5 months had a tendency toward
increased thresholds, although the difference was not significant. Major degeneration of the VIIIth cranial nerve was
excluded by the presence of distortion product otoacoustic
emissions (reflecting cochlear amplification) and morphological examination (not shown). Taken together, auditory
signal propagation was delayed in PLPnull*M6Bnull-mice.
Myelin Protein Composition
To analyze its molecular composition, CNS myelin was
purified from PLPnull*M6Bnull-mice. By immunoblot, the
abundance of most myelin-associated proteins was unaltered, including CNP, MAL, MOG, CD9, plasmolipin, flotillin, SEPT6, and caveolin 1 (Figs. 4F and 6A). In contrast, the abundance of N-cadherin, MAG, OSP/Claudin
11, and CD81 was moderately increased. The abundance
of one M6B splice-isoform (aTMDw) was strongly
increased in PLPnull-myelin (Fig. 4F). The virtual absence
of SIRT2 from PLPnull-myelin has been reported (Werner
et al., 2007). Taken together, no myelin protein was elevated or diminished exclusively in PLPnull*M6Bnull-myelin, suggesting that proteolipid deficiency is directly responsible for the PLPnull*M6Bnull-phenotype. To determine the association of myelin proteins with membrane
microdomains, myelin was extracted with CHAPS and
separated by sucrose density gradient centrifugation. By
immunoblot (Fig. 6B), only MOG and flotillin were slightly
shifted to the soluble fraction while the solubility of most
proteins was unaltered in PLPnull*M6Bnull-myelin.
Altered Myelin Lipid Composition
Absence of proteolipids clearly affected the lipid composition of myelin, as demonstrated by thin layer chromatography (Fig. 6C), a method suitable for identifying
qualitative differences (Lappe-Siefke et al., 2003; Saher
et al., 2005). When HPLC was used to quantify cholesterol levels (Keller et al., 2004), cholesterol was reduced
by about 40% in PLPnull and PLPnull*M6Bnull myelin
compared with M6Bnull and WT myelin (Fig. 6D). This
suggests that PLP, by virtue of its property as an abundant cholesterol-binding protein in oligodendrocytes that
is efficiently incorporated into myelin, mediates the
enrichment of cholesterol in myelin membranes.
Dysmyelination
While purifying CNS myelin for immunoblot analysis
(see above), we noted that that the interphase between
GLIA
0.85 M and 0.32 M sucrose was considerably diminished
for PLPnull*M6Bnull-brains compared to all other genotypes. We therefore hypothesized that the functional
impairments resulted from dysmyelination. We analyzed
myelination in the optic nerve and spinal cord by EM.
In the optic nerve at 14 days, myelin assembly was
almost complete in WT, M6Bnull, and PLPnull-mice (Fig.
7A), but PLPnull*M6Bnull-mice lacked myelin (Fig. 7B,
magnified in Fig. 7E). Also at 1 month (Fig. 7C), myelination was clearly delayed in the optic nerve (Fig. 7D)
and the spinal cord (not shown) when compared with
single mutants. Oligodendrocyte processes were longer
and thicker and were often seen engulfing axons with a
noncompacted cellular process (Fig. 7E,E’). Most largecaliber axons were myelinated, and myelin thickness
appeared normal, although intraperiod lines were partly
condensed as in PLPnull-mice (Klugmann et al., 1997;
Mobius et al., 2008).
Next we analyzed histological sections from brains
and spinal cords of PLPnull*M6Bnull-mice and controls at
2.5 months (n 5 6 per genotype). By Nissl and HE staining, or myelin silver impregnation, the thickness of the
corpus callosum and anterior commissure measured only
two thirds in PLPnull*M6Bnull mice compared with all
control genotypes (Fig. 7F,G), possibly caused by both
developmental delay (dysmyelination) and myelin degeneration (demyelination). In PLPnull*M6Bnull-mice, myelin in the striatal white matter and in spinal cord gray
matter was 86% and 67%, respectively, of the levels in
all other genotypes (Fig. 7H,I). Hypomyelination could
potentially contribute to the observed ventricular
enlargement and slight overall reduction in brain size.
Axonal Swellings, Microglia Activation, and
Astrogliosis
While developmental myelination is not markedly
impaired in PLPnull mice (Klugmann et al., 1997), secondary degeneration affects myelinated axons at older
age (Griffiths et al., 1998). When stained for APP,
M6Bnull and WT mice did not reveal such axonal degeneration in brain and spinal cord (Fig. 8A,B). However, in
double mutants, axonal swellings (Fig. 8D,E) were of
earlier onset and much more frequent than in PLPnullmice (Fig. 8C).
Neurodegeneration in PLPnull*M6Bnull-mice was
accompanied by reactive microgliosis and astrogliosis as
shown by MAC-3 and GFAP-immunostaining (Fig. 8F–
M), but not by B- or T-cell infiltration (not shown). By
caspase staining, we observed apoptotic cells in the CNS
of PLPnull*M6Bnull but not in control mice (Fig. 8N). At
least a subpopulation of apoptotic cells were CNP1 oligodendrocytes with condensed nuclei (not shown). Interestingly, we also detected proliferating (MIB51) cells and a
higher density of CNP1 oligodendrocytes in adult
PLPnull*M6Bnull-mice than in controls (Fig. 8O,P). Generalized, pathology was more prominent in white than
in gray matter and was also observed in the retina and
the PNS (dorsal roots; Fig. 8Q–X).
PROTEOLIPIDS SEQUESTER CHOLESTEROL IN MYELINATION
13
Fig. 6. Myelin protein and lipid analysis. (A): Immunoblot analysis
of myelin (5 lg protein per lane) to test for the presence of the indicated myelin proteins. Note that the abundance of most proteins was
unaltered, while N-Cadherin, MAG, and OSP were increased in PLPnull
and dKO myelin, and SIRT2 was virtually absent. (B): Myelin was
solubilized with CHAPS and floated in a density gradient. Twelve fractions were obtained from the top to the bottom, separated by SDSPAGE, and blotted. The sucrose concentration in each fraction was
measured with a refractometer. The solubility of most myelin proteins
did not depend on proteolipids, while MOG and Flotillin appeared
slightly shifted toward the high-sucrose fractions. (C): Qualitative thinlayer chromatography of myelin lipids extracted from myelin-enriched
brain fractions. The principal lipid composition was unaltered in myelin
purified from mutant compared with control (WT) brains, except for an
apparently reduced abundance of cholesterol (Chol) in PLPnull and
PLPnull*M6Bnull (dKO) myelin (GalC, Galactosylcerebroside; PE, phosphatidyl-ethanolamin; Sulf, sulfatide; PC, phosphatidyl choline; PI,
phosphatidyl-inositol; PS, phosphatidyl-serine; SM, sphingomyelin).
Two biological replicates per genotype are shown. (D): Quantitative
HPLC to determine sterol concentration in myelin (three biological replicates per genotype). Data represent the mean (6 SEM), with WT set
to 100%. One-way ANOVA with Bonferroni’s multiple post-test comparison was performed (*, P<0.05). The cholesterol content was significantly reduced in PLPnull and dKO myelin compared with WT and
M6Bnull myelin.
The physiological relevance of the neurodegenerative
changes was documented by far-field electric potentials
from the occipital region in response to light (Fig. 9). In
4.5-month-old mice, VEP were easily recorded for
M6Bnull and PLPnull-mice. Conversely, no reproducible
responses were detected in PLPnull*M6Bnull-mice.
Indeed, the axonal degeneration profiles in the optic
nerves of PLPnull*M6Bnull-mice at 4.5 months (Fig.
10A,B) structurally resembled those of PLPnull-mice at
20 months (Griffiths et al., 1998) but were more numerous. Macrophages filled with lipid droplets and myelin
debris were very frequent (Fig. 10C,D). It was striking
that the only viable axons were unmyelinated. Taken to-
gether, PLP and M6B serve overlapping functions in
myelination and also in the neuroprotective function of
CNS myelin (Nave, 2010; Yin et al., 2006).
DISCUSSION
We report the intriguing observation that CNS myelin,
which lacks its major transmembrane protein (PLP), also
comprises a strongly reduced cholesterol level. Considering that PLPnull-mice show normal motor-sensory functions in the first year (Klugmann et al., 1997), ‘‘high-cholesterol’’ is thus not indispensible for physiologically
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14
WERNER ET AL.
Fig. 7. Dysmyelination in mice lacking PLP and M6B. (A, B): EM
analysis of optic nerves at 14 days. In M6Bnull mice, both large and
small diameter axons were myelinated (arrowheads in (A), as in WT
and PLPnull controls. In contrast, no myelin was present in
PLPnull*M6Bnull (dKO) optic nerves (B) at this age. (C, D): Examination
of the optic nerve at 28 days of age reveals complete myelination in
M6Bnull, (C) as in WT and PLPnull controls (not shown). In dKO littermates (D), most small diameter axons lacked compact myelin while
larger diameter axons were myelinated with sheaths of apparently
appropriate thickness. (E, E0 ): In dKO white matter tracts, many axonoligodendrocyte units were found at a stage where the axon (blue in
the false color image in (E0 ) is surrounded by one layer of oligodendroglial process (red in E0 ), apparently halted at an engulfed premyelin
stage. The representative example is from the optic nerve at 14 days of
age. (F, G): Brain sections of control (F) and PLPnull*M6Bnull mice
GLIA
(dKO) (G) were silver-impregnated to visualize myelin (age 2.5 months).
Note enlarged ventricles (in G), possibly caused by white matter atrophy. The corpus callosum (CC) and anterior commissure (AC) were
diminished by approximately one third in dKO. Hypomyelination was
also present in the dKO cortex (Ctx). (H, I): CNS sections were subjected to CNP IHC to quantify myelin in the striatum or spinal cord
gray matter (six animals per genotype, three sections per animal). Microscopic images (203) were imported into Scion Image software and
striatum or spinal cord gray matter selected in freehand mode. CNP1
area units were calculated as percentage of the selected area. Given is
the mean (6 SEM). Unpaired t-test was performed and two-tailed Pvalues were calculated comparing each group to the WT control (****,
P<0.0001). [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
Fig. 8. Neuropathology in proteolipid mutants. Brain sections from
mice of the indicated genotypes were subjected to IHC (age 2.5 months).
Representative examples from the lateral corpus callosum (CC) are shown
(A–D, F–O). Specific labeling is in brown, and nuclei were counterstained
(blue). (A–D): APP immunolabeling was used as a marker for axonal swellings, which represent an early stage of neurodegeneration. In both WT
and M6Bnull, APP labeling was restricted to and weak in neuronal cell
bodies. Additional labeling of axonal swellings was rare in PLPnull white
matter at this age but very frequent in both white and gray matter of
PLPnull*M6Bnull (dKO) (enlarged in inset in (D)). (E): EM of an axonal
swelling in a longitudinal section of the optic nerve in dKO. (F–I): Activated microglia were detected by MAC3 immunolabeling. Except for the
choroid plexus, MAC31 cells were very infrequent in the CNS of WT or
M6Bnull animals. In contrast, MAC31 cells were detected rarely in PLPnull
and frequently in dKO, suggesting extensive microglia activation
(enlarged in inset in I). (J–M): Numbers of GFAP1 astrocytes were estimated WT<M6Bnull<PLPnull<dKO, suggesting extensive astrogliosis in
dKO compared with single mutants. (N): Apoptotic CM11 cells were identified by immunolabeling, detecting activated caspase. Apoptosis was
obvious in dKO white matter but not detectable in controls. (O): Dividing
MIB51 cells were present in the CC of dKO but not in controls. (P): To
quantify relative oligodendrocyte numbers, we counted CNP1 cell bodies
in spinal cord gray matter (three nonconsequtive sections per animal, six
animals per genotype). Data represent the mean (6 SEM). An ANOVA
unpaired two-tailed t-test was performed (**, P<0.01; ***, P<0.005). Oligodendrocyte density was significantly increased in dKO compared with WT
controls. Secondary neuropathology was also found in the peripheral nervous system (dorsal roots, (Q–T) and the eye (U–X)). Note that GFAP1 cells
were detected in dKO but not WT, M6Bnull or PLPnull dorsal roots. In the
eye of WT or M6Bnull mice, GFAP expression was restricted to astrocytes
in the ganglion cell layer (GCL). Conversely, strong GFAP expression was
found in presumptive M€
uller glia of the nuclear layer (NL) in dKO, and
some in PLPnull. PE, pigment epithelium. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
16
WERNER ET AL.
Overlapping But Nonidentical Functions of
Myelin Proteolipids
Fig. 9. Visual system function in proteolipid mutants. VEP in
response to light flashes were determined at the age of 19–25 weeks.
Animal numbers were 6 M6Bnull, 8 PLPnull, and 5 PLPnull*M6Bnull. The
Figure displays the grand average (in dark color) in voltage with SEM
(in light color). Mean amplitudes were 12.9 (6 3.5) lV for M6Bnull, and
6.1 (6 1.2) lV for PLPnull mice, and latencies were 36.1 (61) ms and
38.67 (61.7) ms, respectively. Absence of measurable VEP suggests that
PLPnull*M6Bnull (dKO) were functionally blind. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
functional myelin. However, PLP emerges as the mediator
for oligodendrocytes to ‘‘enrich’’ cholesterol to the high
level present in normal myelin by molecular association
and cotransport. Moreover, we show that PLP has a genuine function in myelination, which in earlier studies was
masked by the presence of its homolog M6B. M6B shares
the cholesterol-binding characteristics of PLP, suggesting
that both proteolipids assist the formation of cholesterolrich membrane during myelination.
The lethal phenotypes of natural PLP1-mutations in
mouse and man are best explained by toxic gain-of-function (Duncan, 2005; Garbern, 2007; Miller et al., 2003),
but the genuine function of PLP has remained elusive
for a long time. Upon the analysis of PLPnull-mice, PLP
emerged as a ‘‘molecular strut’’’ to stabilize compact
myelin at the intraperiod lines (Mobius et al., 2008).
Moreover, PLPnull-mice display degenerating myelinated
axons (Edgar et al., 2004; Griffiths et al., 1998),
although the molecular mechanisms are unclear.
Recently, we found that PLP deficiency causes secondary
changes in the myelin proteome that may contribute to
axonal degeneration (Werner et al., 2007). This manuscript reveals that the functions of PLP and M6B in developmental myelination overlap, as PLPnull*M6Bnulldouble mutants remain severely dysmyelinated, in
marked contrast to either single mutant. Their phenotype was analyzed from the molecular to the systems
level.
GLIA
M6B is a structural homolog of the DM20 splice isoform
encoded by the Plp1 gene. Both proteolipids share >50%
sequence identity, but they differ in many respects. For
example, the Gpm6b gene has a complex pattern of transcription initiation and RNA splicing, thus encoding eight
protein isoforms with different N- and C-termini (Schweitzer
et al., 2006). While PLP expression is strongly enriched in
oligodendrocytes, M6B is found in both oligodendrocytes and
neurons (Yan et al., 1993) and at low level in many tissues
(Werner et al., 2001). Mutations affecting the human PLP1
gene cause Pelizaeus-Merzbacher disease (Duncan, 2005;
Garbern, 2007). Conversely, no disease has been linked to
GPM6B, while altered M6B-expression was reported for autism (Purcell et al., 2001), amyotrophic lateral sclerosis
(Dangond et al., 2004), glioblastoma (Castells et al., 2010),
and suicide-completors (Fiori et al., 2011), although no causative involvement in the pathophysiology of oligodendrocytes
or other cells has been reported. M6Bnull-mice display normal myelination, motor development, long-term axonal integrity, and only minor distortions at the myelin-axon interface. Conversely, PLP is required for the normal ultrastructure of compact CNS myelin (at the intraperiod lines) and
affects the abundance of other myelin proteins (e.g., SIRT2)
(Werner et al., 2007). By mass-spectrometric quantification,
PLP is much more abundant than M6B (15.4% versus
0.05% of total myelin protein). Taken together, the contributions of PLP and M6B to myelin are fairly distinct.
In contrast, the expression of either PLP or M6B is sufficient to allow complete myelination. We noted the
increased abundance in PLPnull-myelin of the M6B-splice
isoform that displays a C-terminus most similar to that of
PLP/DM20. However, by immunoblotting the amount of
total M6B in PLPnull-myelin is only doubled compared
with WT-myelin and thus far from physically replacing
PLP. The functional overlap of the two proteolipids, which
display such a different abundance in myelin, suggests
they are not essential as structural myelin proteins but
that their critical ‘‘common function’’ is served in an intracellular compartment in which they also colocalize. Considering their association with membrane-cholesterol
(Simons et al., 2000; this study), we propose that both proteolipids accumulate cholesterol in the oligodendroglial secretory pathway for the formation of cholesterol-rich
membrane domains required for efficient trafficking to the
growing myelin sheath. We note that also other proteins,
which are involved in intracellular trafficking of myelin
constituents, can be found as low-abundance myelin proteins. For example, VAMP2, VAMP3, SNAP23 (Feldmann
et al., 2009), and cdc42 (Thurnherr et al., 2006) were
detected as ‘‘trapped’’ in the myelin compartment.
Association and Cotransport of Myelin Proteins
and Lipids
During myelination, a large amount of membrane
must be assembled and sorted in short time. It has been
PROTEOLIPIDS SEQUESTER CHOLESTEROL IN MYELINATION
17
Fig. 10. Demyelination in adult mice lacking both PLP and M6B.
(A): Toluidin-stained semithin sections from M6Bnull mice at 4.5 months
of age were analyzed. The optic nerve was completely myelinated with
no signs of degeneration, as in WT or PLPnull controls. (B): No myelin
was detectable in the PLPnull*M6Bnull (dKO) optic nerve at this age.
(C, D): The degree of degeneration in dKO optic nerves is revealed
by EM. Some unmyelinated small-diameter axons were detectable
(arrowheads in C), but many axons were degenerated (*, axonal swelling in C). Myelin remnants were found undigested within macrophages
(** in D). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
suggested that myelin lipids and proteins control each
other’s trafficking in the oligodendroglial secretory pathway (Simons and Trotter, 2007). Indeed, the functions of
abundant myelin lipids (e.g., glycosphingolipids, cholesterol) begin to be revealed. For example, the myelin-tetraspan MAL organizes apical membrane regions, at least in
vitro (Magal et al., 2009; Ramnarayanan and Tuma, 2011).
Trafficking of MAL and hence its abundance in mature
myelin depend on the associated glycosphingolipid 3-OSulfogalactosylceramide (SGalC), while the lack of MAL or
SGalC cause similar abnormalities of paranodal myelin
(Saravanan et al., 2004; Schaeren-Wiemers et al., 2004).
High-level cholesterol is critical for normal myelination,
as demonstrated by the dysmyelinated phenotype of conditional mouse mutants in which myelinating glia lack squalene synthase (SQS) and thus cholesterol biosynthesis
(Saher et al., 2005, 2009). However, CNS myelination is
only delayed in SQS mutants, while the protein composition
of myelin is not obviously altered, with the molar PLP-tocholesterol ratio maintained. Thus, in the absence of oligo-
dendroglial cholesterol biosynthesis, cholesterol uptake
from neighboring WT cells is possible and ultimately rate
limiting for myelin biogenesis. The molecular mechanism
by which membrane cholesterol is enriched in SQS-deficient
oligodendrocytes is obviously intact in SQS mutant mice,
demonstrating that cholesterol synthesis in oligodendrocytes by itself is not sufficient to achieve the ‘‘high-cholesterol’’ content of normal myelin. The relevance of the molar
PLP-to-cholesterol ratio for trafficking into myelin was
recently supported in PLP-overexpressing transgenic mice,
a model of Pelizaeus-Merzbacher disease (Werner et al.,
1998). In these mice, excess PLP accumulates in oligodendroglial late endosomes/lysosomes, and the addition of supplementary cholesterol was therapeutic by facilitating the
incorporation of PLP into myelin (Saher et al., 2012).
In this study, we show that myelin purified from
PLPnull or dysmyelinated PLPnull*M6Bnull-mice exhibits
a strongly reduced cholesterol content (240%), comparable to cholesterol levels in other plasma membranes
(Morell and Jurevics, 1996). Notably, the rate of brain
GLIA
18
WERNER ET AL.
enriching proteins exist. We speculate that other myelin-tetraspans may partly compensate for proteolipid
deficiency. For example, tetraspanins (CD9, CD81,
CD82, TSPAN2), plasmolipin, and MAL are present in
CNS myelin (Birling et al., 1999; Bosse et al., 2003;
Kagawa et al., 1997; Mela and Goldman, 2009; Schaeren-Wiemers et al., 2004; Terada et al., 2002). At least
some of these indeed associate with cholesterol (Charrin
et al., 2003; Claas et al., 2001). Taken together, small
hydrophobic tetraspans may overlap in the assembly
and cotransport of membrane lipids and thus contribute
to the 5–10-fold enrichment of cholesterol in plasma
membranes compared with the endoplasmic reticulum
(van Meer et al., 2008) and its even higher concentration
in myelin. However, functional differences between particular tetraspans appear at least as relevant, as illustrated by the specific structural consequences of their
deficiency in mutant mice.
Coevolution of Myelin Protein and Lipid
Composition
Fig. 11. Model of proteolipid-dependent myelination. Proteolipid protein PLP and its homolog glycoprotein M6B sequester cholesterol in the secretory pathway of oligodendrocytes, yielding membranes of high cholesterol content. Either of the two proteolipids can mediate efficient developmental myelination, suggesting partial functional redundancy in the
trafficking of myelin constituents through the secretory pathway of oligodendrocytes. PLP is incorporated into myelin efficiently, thereby determining the cholesterol content of mature myelin by co-transport. Conversely,
M6B does not measurably contribute to the cholesterol content of myelin
because its abundance in myelin is >200-fold below that of PLP. M6B is
trapped in the myelin compartment at low abundance, similar to other
proteins involved in the trafficking of prospective myelin membrane such
as VAMP2, VAMP3, SNAP23, and cdc42. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
cholesterol synthesis does not differ between PLPnull and
WT-mice (Jurevics et al., 2003). Thus, oligodendrocytes
require PLP to achieve the high cholesterol level in myelin, indicating that proteolipids and cholesterol indeed
control each other’s trafficking in the oligodendroglial secretory pathway. However, myelination is not markedly
impaired in PLPnull-mice (Klugmann et al., 1997), which
exhibit the same myelin–cholesterol content as dysmyelinated PLPnull*M6Bnull-mice, while M6B-deficiency by
itself does not measurably affect myelin-cholesterol.
Thus, we propose that the cellular compartment of oligodendrocytes that requires cholesterol enrichment is not
the mature myelin sheath but the secretory pathway. For
example, efficient vesicle budding and fusion could
depend on cholesterol by affecting membrane curvature
(Huttner and Zimmerberg, 2001). Interestingly, the analysis of other vesicle preparations has revealed a high cholesterol content (Takamori et al., 2006).
Limited myelination is possible also in PLPnull
*M6Bnull-mice, suggesting that further cholesterolGLIA
In evolution, proteolipids emerged before myelin
(Mobius et al., 2008). PLP is of low abundance in fish
but became the major CNS myelin protein in tetrapods,
coinciding with the evolutionary emergence of the PLPspecific intracellular loop (Yoshida and Colman, 1996).
This domain enhances the efficient incorporation of PLP
into myelin (Trapp et al., 1997; Wang et al., 2008) and
thus may constitute a sorting signal. Indeed, M6B colocalizes with PLP in the oligodendroglial secretory pathway but only PLP is efficiently incorporated into myelin.
Thus, at the fish-to-tetrapod transition PLP may have
been recruited from its function in intracellular trafficking as the most abundant CNS myelin protein. Indeed,
the high cholesterol level in myelin may have evolved by
its molecular association with PLP. This idea is supported by the finding that the cholesterol content of
myelin is similar across various tetrapod species (Burgisser et al., 1986) but lower in fish (Kirschner et al.,
1989). Together this suggests an intriguing correlation
between the evolutionarily increased abundance of PLP
and of cholesterol in CNS myelin at the root of tetrapods, coinciding with the emergence of the PLP-specific
loop.
CONCLUSIONS
Taken together, PLP determines the high cholesterol
content of CNS myelin by molecular association and cotransport, using the PLP-specific myelin-targeting signal. In contrast, M6B is not efficiently incorporated into
myelin, and its low abundance is not sufficient to measurably contribute to the cholesterol level of myelin.
More speculatively, we suggest a model for the function
of both proteolipids in enriching cholesterol locally in
the oligodendroglial secretory pathway (schematically
depicted in Fig. 11). Here, at least one proteolipid (PLP
PROTEOLIPIDS SEQUESTER CHOLESTEROL IN MYELINATION
or M6B) is required for efficient trafficking and thus
normal myelination. We propose that the failure to efficiently traffic prospective myelin membrane through the
secretory pathway represents a plausible cause of dysmyelination in mice lacking both proteolipids.
ACKNOWLEDGMENTS
We thank A. Fahrenholz and T. Ruhwedel for technical assistance, and J.M. Edgar, O. Jahn, J. Patzig, S.
Forss-Petter, H. Lassmann, S. Papiol, and M. Simons for
discussions. Antibodies were kindly provided by F.
Bosse, P.J. Brophy, C. Linington, and N. SchaerenWiemers, photo-activatable cholesterol by C. Thiele, and
plasmids by A. Dhaunchak, F. Varoqueaux, and N.
Brose.
REFERENCES
Birling MC, Tait S, Hardy RJ, Brophy PJ. 1999. A novel rat tetraspan
protein in cells of the oligodendrocyte lineage. J Neurochem 73:2600–
2608.
Bosse F, Hasse B, Pippirs U, Greiner-Petter R, Muller HW. 2003. Proteolipid plasmolipin: Localization in polarized cells, regulated expression and lipid raft association in CNS and PNS myelin. J Neurochem
86:508–518.
Burgisser P, Matthieu JM, Jeserich G, Waehneldt TV. 1986. Myelin lipids: A phylogenetic study. Neurochem Res 11:1261–1272.
Buser AM, Erne B, Werner HB, Nave KA, Schaeren-Wiemers N. 2009.
The septin cytoskeleton in myelinating glia. Mol Cell Neurosci
40:156–166.
Cammer W, Fredman T, Rose AL, Norton WT. 1976. Brain carbonic
anhydrase: Activity in isolated myelin and the effect of hexachlorophene. J Neurochem 27:165–171.
Castells X, Acebes JJ, Boluda S, Moreno-Torres A, Pujol J, Julia-Sape M,
Candiota AP, Arino J, Barcelo A, Arus C. 2010. Development of a predictor for human brain tumors based on gene expression values
obtained from two types of microarray technologies. Omics 14:157–164.
Charrin S, Manie S, Thiele C, Billard M, Gerlier D, Boucheix C, Rubinstein E. 2003. A physical and functional link between cholesterol and
tetraspanins. Eur J Immunol 33:2479–2489.
Chrast R, Saher G, Nave KA, Verheijen MH. 2011. Lipid metabolism in
myelinating glial cells: Lessons from human inherited disorders and
mouse models. J Lipid Res 52:419–434.
Claas C, Stipp CS, Hemler ME. 2001. Evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid
rafts. J Biol Chem 276:7974–7984.
Dangond F, Hwang D, Camelo S, Pasinelli P, Frosch MP, Stephanopoulos G, Brown RH Jr., Gullans SR. 2004. Molecular signature of latestage human ALS revealed by expression profiling of postmortem spinal cord gray matter. Physiol Genomics 16:229–239.
Dhaunchak AS, Nave KA. 2007. A common mechanism of PLP/DM20
misfolding causes cysteine-mediated endoplasmic reticulum retention
in oligodendrocytes and Pelizaeus-Merzbacher disease. Proc Natl
Acad Sci USA 104:17813–17818.
Duncan ID. 2005. The PLP mutants from mouse to man. J Neurol Sci
228:204–205.
Edgar JM, McLaughlin M, Werner HB, McCulloch MC, Barrie JA,
Brown A, Faichney AB, Snaidero N, Nave KA, Griffiths IR. 2009.
Early ultrastructural defects of axons and axon-glia junctions in mice
lacking expression of Cnp1. Glia 57:1815–1824.
Edgar JM, McLaughlin M, Yool D, Zhang SC, Fowler JH, Montague P,
Barrie JA, McCulloch MC, Duncan ID, Garbern J, Nave KA, Griffiths
IR. 2004. Oligodendroglial modulation of fast axonal transport in a
mouse model of hereditary spastic paraplegia. J Cell Biol 166:
121–131.
Eiberger J, Kibschull M, Strenzke N, Schober A, Bussow H, Wessig C,
Djahed S, Reucher H, Koch DA, Lautermann J, Moser T, Winterhager E, Willecke K. 2006. Expression pattern and functional characterization of connexin29 in transgenic mice. Glia 53:601–611.
Epand RM. 2006. Cholesterol and the interaction of proteins with membrane domains. Prog Lipid Res 45:279–294.
19
Feldmann A, Winterstein C, White R, Trotter J, Kramer-Albers EM.
2009. Comprehensive analysis of expression, subcellular localization,
and cognate pairing of SNARE proteins in oligodendrocytes. J Neurosci Res 87:1760–1772.
Fiori LM, Zouk H, Himmelman C, Turecki G. 2011. X chromosome and
suicide. Mol Psychiatry 16:216–226.
Garbern JY. 2007. Pelizaeus-Merzbacher disease: Genetic and cellular
pathogenesis. Cell Mol Life Sci 64:50–65.
Gielen E, Baron W, Vandeven M, Steels P, Hoekstra D, Ameloot M.
2006. Rafts in oligodendrocytes: Evidence and structure–function
relationship. Glia 54:499–512.
Gitik M, Liraz-Zaltsman S, Oldenborg PA, Reichert F, Rotshenker S.
2011. Myelin down-regulates myelin phagocytosis by microglia and
macrophages through interactions between CD47 on myelin and SIRPalpha (signal regulatory protein-alpha) on phagocytes. J Neuroinflammation 8:24.
Gow A, Southwood CM, Li JS, Pariali M, Riordan GP, Brodie SE, Danias J, Bronstein JM, Kachar B, Lazzarini RA. 1999. CNS myelin and
sertoli cell tight junction strands are absent in Osp/claudin-11 null
mice. Cell 99:649–659.
Griffiths I, Klugmann M, Anderson T, Yool D, Thomson C, Schwab MH,
Schneider A, Zimmermann F, McCulloch M, Nadon N, Nave KA.
1998. Axonal swellings and degeneration in mice lacking the major
proteolipid of myelin. Science 280:1610–1613.
Huttner WB, Zimmerberg J. 2001. Implications of lipid microdomains
for membrane curvature, budding and fission. Curr Opin Cell Biol
13:478–484.
Jahn O, Tenzer S, Werner HB. 2009. Myelin proteomics: Molecular
anatomy of an insulating sheath. Mol Neurobiol 40:55–72.
Johns TG, Bernard CC. 1999. The structure and function of myelin oligodendrocyte glycoprotein. J Neurochem 72:1–9.
Jung M, Sommer I, Schachner M, Nave KA. 1996. Monoclonal antibody
O10 defines a conformationally sensitive cell-surface epitope of proteolipid protein (PLP): Evidence that PLP misfolding underlies dysmyelination in mutant mice. J Neurosci 16:7920–7929.
Jung M, Crang AJ, Blakemore WF, Hoppe D, Kettenmann H, Trotter J.
1994. In vitro and in vivo characterisation of glial cells immortalised
with a temperature sensitive SV40 T antigen-containing retrovirus. J
Neurosci Res 37:182–196.
Jurevics H, Hostettler J, Sammond DW, Nave KA, Toews AD, Morell P.
2003. Normal metabolism but different physical properties of myelin
from mice deficient in proteolipid protein. J Neurosci Res 71:826–834.
Kagawa T, Mekada E, Shishido Y, Ikenaka K. 1997. Immune systemrelated CD9 is expressed in mouse central nervous system myelin at
a very late stage of myelination. J Neurosci Res 50:312–320.
Keller RK, Small M, Fliesler SJ. 2004. Enzyme blockade: A nonradioactive method to determine the absolute rate of cholesterol synthesis in
the brain. J Lipid Res 45:1952–1957.
Kirschner DA, Inouye H, Ganser AL, Mann V. 1989. Myelin membrane
structure and composition correlated: A phylogenetic study. J Neurochem 53:1599–1609.
Klugmann M, Schwab MH, Puhlhofer A, Schneider A, Zimmermann F,
Griffiths IR, Nave KA. 1997. Assembly of CNS myelin in the absence
of proteolipid protein. Neuron 18:59–70.
Kramer EM, Schardt A, Nave KA. 2001. Membrane traffic in myelinating oligodendrocytes. Microsc Res Technol 52:656–671.
Kr€
amer-Albers E-M, Bretz N, Tenzer S, Winterstein C, M€
obius W,
Berger H, Nave K-A, Schild H, Trotter J. 2007. Oligodendrocytes
secrete exosomes containing major myelin and stress-protective proteins: Trophic support for axons? Proteomics Clin Appl 1:1446–1461.
Kr€
amer-Albers EM, Gehrig-Burger K, Thiele C, Trotter J, Nave KA.
2006. Perturbed interactions of mutant proteolipid protein/DM20
with cholesterol and lipid rafts in oligodendroglia: Implications for
dysmyelination in spastic paraplegia. J Neurosci 26:11743–11752.
Lappe-Siefke C, Goebbels S, Gravel M, Nicksch E, Lee J, Braun PE,
Griffiths IR, Nave KA. 2003. Disruption of Cnp1 uncouples oligodendroglial functions in axonal support and myelination. Nat Genet
33:366–374.
Magal LG, Yaffe Y, Shepshelovich J, Aranda JF, de Marco Mdel C,
Gaus K, Alonso MA, Hirschberg K. 2009. Clustering and lateral concentration of raft lipids by the MAL protein. Mol Biol Cell 20:3751–
3762.
Maxfield FR, van Meer G. 2010. Cholesterol, the central lipid of mammalian cells. Curr Opin Cell Biol 22:422–429.
Mela A, Goldman JE. 2009. The tetraspanin KAI1/CD82 is expressed
by late-lineage oligodendrocyte precursors and may function to
restrict precursor migration and promote oligodendrocyte differentiation and myelination. J Neurosci 29:11172–11181.
Miller MJ, Haxhiu MA, Georgiadis P, Gudz TI, Kangas CD, Macklin
WB. 2003. Proteolipid protein gene mutation induces altered ventilatory response to hypoxia in the myelin-deficient rat. J Neurosci
23:2265–2273.
GLIA
20
WERNER ET AL.
Mimault C, Giraud G, Courtois V, Cailloux F, Boire JY, Dastugue B,
Boespflug-Tanguy O. 1999. Proteolipoprotein gene analysis in 82
patients with sporadic Pelizaeus-Merzbacher Disease: Duplications,
the major cause of the disease, originate more frequently in male
germ cells, but point mutations do not. The Clinical European Network on Brain Dysmyelinating Disease. Am J Hum Genet 65:
360–369.
Mobius W, Patzig J, Nave KA, Werner HB. 2008. Phylogeny of proteolipid proteins: divergence, constraints, and the evolution of novel functions in myelination and neuroprotection. Neuron Glia Biol 4:
111–127.
Morell P, Jurevics H. 1996. Origin of cholesterol in myelin. Neurochem
Res 21:463–470.
Nave KA. 2010. Myelination and support of axonal integrity by glia.
Nature 468:244–252.
Nawaz S, Kippert A, Saab AS, Werner HB, Lang T, Nave KA, Simons
M. 2009. Phosphatidylinositol 4,5-bisphosphate-dependent interaction
of myelin basic protein with the plasma membrane in oligodendroglial cells and its rapid perturbation by elevated calcium. J Neurosci
29:4794–4807.
Norton WT, Poduslo SE. 1973. Myelination in rat brain: Changes in
myelin composition during brain maturation. J Neurochem 21:
759–773.
O’Brien JS. 1965. Stability of the Myelin Membrane. Science 147:
1099–1107.
Patzig J, Jahn O, Tenzer S, Wichert SP, de Monasterio-Schrader P,
Rosfa S, Kuharev J, Yan K, Bormuth I, Bremer J, Aguzzi A, Orfaniotou F, Hesse D, Schwab MH, Mobius W, Nave KA, Werner HB. 2011.
Quantitative and integrative proteome analysis of peripheral nerve
myelin identifies novel myelin proteins and candidate neuropathy
loci. J Neurosci 31:16369–16386.
Purcell AE, Jeon OH, Zimmerman AW, Blue ME, Pevsner J. 2001. Postmortem brain abnormalities of the glutamate neurotransmitter system in autism. Neurology 57:1618–1628.
Quarles RH. 2007. Myelin-associated glycoprotein (MAG): Past, present
and beyond. J Neurochem 100:1431–1448.
Ramnarayanan SP, Tuma PL. 2011. MAL, but not MAL2, expression
promotes the formation of cholesterol-dependent membrane domains
that recruit apical proteins. Biochem J 439:497–504.
Saher G, Quintes S, Mobius W, Wehr MC, Kramer-Albers EM, Brugger
B, Nave KA. 2009. Cholesterol regulates the endoplasmic reticulum
exit of the major membrane protein P0 required for peripheral myelin compaction. J Neurosci 29:6094–6104.
Saher G, Brugger B, Lappe-Siefke C, Mobius W, Tozawa R, Wehr MC,
Wieland F, Ishibashi S, Nave KA. 2005. High cholesterol level is
essential for myelin membrane growth. Nat Neurosci 8:468–475.
Saher G, Rudolphi F, Corthals K, Ruhwedel T, Schmidt KF, Lowel S,
Dibaj P, Barrette B, Mobius W, Nave KA. 2012. Therapy of PelizaeusMerzbacher disease in mice by feeding a cholesterol-enriched diet.
Nat Med 18:1130–1135.
Saravanan K, Schaeren-Wiemers N, Klein D, Sandhoff R, Schwarz A,
Yaghootfam A, Gieselmann V, Franken S. 2004. Specific downregulation and mistargeting of the lipid raft-associated protein MAL in a
glycolipid storage disorder. Neurobiol Dis 16:396–406.
Schaeren-Wiemers N, Bonnet A, Erb M, Erne B, Bartsch U, Kern F,
Mantei N, Sherman D, Suter U. 2004. The raft-associated protein
MAL is required for maintenance of proper axon–glia interactions in
the central nervous system. J Cell Biol 166:731–742.
Schweitzer J, Becker T, Schachner M, Nave KA, Werner H. 2006. Evolution of myelin proteolipid proteins: gene duplication in teleosts and
GLIA
expression pattern divergence. Mol Cell Neurosci 31:161–177.
Simons K, Ikonen E. 1997. Functional rafts in cell membranes. Nature
387:569–572.
Simons M, Trotter J. 2007. Wrapping it up: the cell biology of myelination. Curr Opin Neurobiol 17:533–540.
Simons M, Kramer EM, Thiele C, Stoffel W, Trotter J. 2000. Assembly
of myelin by association of proteolipid protein with cholesterol- and
galactosylceramide-rich membrane domains. J Cell Biol 151:143–154.
Sons MS, Busche N, Strenzke N, Moser T, Ernsberger U, Mooren FC,
Zhang W, Ahmad M, Steffens H, Schomburg ED, Plomp JJ, Missler
M. 2006. alpha-Neurexins are required for efficient transmitter
release and synaptic homeostasis at the mouse neuromuscular junction. Neuroscience 138:433–446.
Takamori S, Holt M, Stenius K, Lemke EA, Grønborg M, Riedel D,
Urlaub H, Schenck S, Br€
ugger B, Ringler P, M€
uller SA, Rammner B,
Gr€
ater F, Hub JS, De Groot BL, Mieskes G, Moriyama Y, Klingauf J,
Grubm€
uller H, Heuser J, Wieland F, Jahn R. 2006. Molecular anatomy of a trafficking organelle. Cell 127:831–846.
Terada N, Baracskay K, Kinter M, Melrose S, Brophy PJ, Boucheix C,
Bjartmar C, Kidd G, Trapp BD. 2002. The tetraspanin protein, CD9,
is expressed by progenitor cells committed to oligodendrogenesis and
is linked to beta1 integrin, CD81, and Tspan-2. Glia 40:350–359.
Thiele C, Hannah MJ, Fahrenholz F, Huttner WB. 2000. Cholesterol
binds to synaptophysin and is required for biogenesis of synaptic
vesicles. Nat Cell Biol 2:42–49.
Thurnherr T, Benninger Y, Wu X, Chrostek A, Krause SM, Nave KA,
Franklin RJ, Brakebusch C, Suter U, Relvas JB. 2006. Cdc42 and Rac1
signaling are both required for and act synergistically in the correct formation of myelin sheaths in the CNS. J Neurosci 26:10110–10119.
Trapp BD, Nishiyama A, Cheng D, Macklin W. 1997. Differentiation
and death of premyelinating oligodendrocytes in developing rodent
brain. J Cell Biol 137:459–468.
van Meer G, Voelker DR, Feigenson GW. 2008. Membrane lipids: where
they are and how they behave. Nat Rev Mol Cell Biol 9:112–124.
Waheed AA, Shimada Y, Heijnen HF, Nakamura M, Inomata M, Hayashi M, Iwashita S, Slot JW, Ohno-Iwashita Y. 2001. Selective binding
of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts). Proc Natl Acad Sci USA 98:4926–4931.
Wang E, Dimova N, Sperle K, Huang Z, Lock L, McCulloch MC, Edgar
JM, Hobson GM, Cambi F. 2008. Deletion of a splicing enhancer disrupts PLP1/DM20 ratio and myelin stability. Exp Neurol 214:322–330.
Werner H, Dimou L, Klugmann M, Pfeiffer S, Nave KA. 2001. Multiple
splice isoforms of proteolipid M6B in neurons and oligodendrocytes.
Mol Cell Neurosci 18:593–605.
Werner H, Jung M, Klugmann M, Sereda M, Griffiths IR, Nave KA.
1998. Mouse models of myelin diseases. Brain Pathol 8:771–793.
Werner HB, Kuhlmann K, Shen S, Uecker M, Schardt A, Dimova K,
Orfaniotou F, Dhaunchak A, Brinkmann BG, Mobius W, Guarente L,
Casaccia-Bonnefil P, Jahn O, Nave KA. 2007. Proteolipid protein is
required for transport of sirtuin 2 into CNS myelin. J Neurosci
27:7717–7730.
Yan Y, Lagenaur C, Narayanan V. 1993. Molecular cloning of M6: Identification of a PLP/DM20 gene family. Neuron 11:423–431.
Yin X, Baek RC, Kirschner DA, Peterson A, Fujii Y, Nave KA, Macklin
WB, Trapp BD. 2006. Evolution of a neuroprotective function of central nervous system myelin. J Cell Biol 172:469–478.
Yoshida M, Colman DR. 1996. Parallel evolution and coexpression of
the proteolipid proteins and protein zero in vertebrate myelin. Neuron 16:1115–1126.