Systematic approaches to central nervous system myelin

Patricia de Monasterio-Schrader1, Olaf Jahn2,3, Stefan Tenzer4, Sven P. Wichert1, Julia Patzig1, and Hauke B. Werner1*

This is the author’s version as accepted for publication. The copyrighted e-offprint is available from Springer Basel AG, as published in Cellular and Molecular Life Sciences (2012), Digital Object Identifier (DOI) 10.1007/s00018-012-0958-9. Authors addresses 1 Department of Neurogenetics

Max Planck Institute of Experimental Medicine, Göttingen, Germany 2 Proteomics Group

Max Planck Institute of Experimental Medicine, Göttingen, Germany 3 DFG Research Center for Molecular Physiology of the Brain Göttingen, Germany 4 Institute of Immunology University Medical Center of the Johannes Gutenberg University Mainz, Germany * Corresponding author: Dr. Hauke Werner Max Planck Institute of Experimental Medicine Department of Neurogenetics Hermann-Rein-Str. 3 D-37075 Göttingen, Germany Tel.: +49 (551) 389-9759 Fax.: +49 (551) 389-9758 E-mail: hauke@em.mpg.de Running title Systematic approaches to CNS myelin Manuscript organization 22 pages, 4 figures, 2 tables, 1 supplemental table Acknowledgements We thank W. Möbius for providing the electron micrograph in Figure 1, J. M. Edgar for critical reading of the manuscript, C. M. Kassmann for discussion and K.-A. Nave for continuous support. ST is supported by the Deutsche Forschungsgemeinschaft (SFB 490 Z3) and the Forschungszentrum Immunologie (FZI) at the University of Mainz, and HBW is supported by the BMBF (DLR-Leukonet) and the European Commission (FP7-LeukoTreat).

Abstract

Rapid signal propagation along vertebrate axons is facilitated by their insulation with myelin, a

plasma membrane specialization of glial cells. The recent application of ‘omics’ approaches to

the myelinating cells of the central nervous system, oligodendrocytes, revealed their mRNA

signatures, enhanced our understanding of how myelination is regulated and established that

the protein composition of myelin is much more complex than previously thought. This

review provides a meta-analysis of the >1200 proteins thus far identified by mass

spectrometry in biochemically purified central nervous system myelin. Contaminating proteins

are surprisingly infrequent according to bioinformatic prediction of subcellular localization and

comparison with the transcriptional profile of oligodendrocytes. The integration of datasets

also allowed the subcategorization of the myelin proteome into functional groups comprising

genes that are coregulated during oligodendroglial differentiation. An unexpectedly large

number of myelin-related genes cause - when mutated in humans - hereditary diseases

affecting the physiology of the white matter. Systematic approaches to oligodendrocytes

and myelin thus provide valuable resources for the molecular dissection of developmental

myelination, glia-axonal interactions, leukodystrophies and demyelinating diseases.

Keywords

Oligodendrocyte; myelin; differentiation; proteome; transcriptome; leukodystrophy;

schizophrenia; multiple sclerosis

Abbreviations

CNS, central nervous system

Ig-CAM, immunoglobulin-like cell adhesion molecule

MBP, myelin basic protein

mOL, myelinating oligodendrocyte

MS, mass spectrometry / mass spectrometric

OPC, oligodendrocyte precursor cell

P0, myelin protein zero

PLP, proteolipid protein

PNS, peripheral nervous system

Introduction

Fast signal propagation along vertebrate axons requires the restriction of action potentials to

short axonal segments termed the nodes of Ranvier. This is achieved by the insulation of the

long segments between the nodes (‘internodes’) with a sleeve of membranous structure

termed myelin [1, 2]. Myelin is a specialization of the plasma membrane of glial cells, which is

organized as multiple, spiral and compacted layers (Fig. 1A). The non-compacted adaxonal

(innermost) myelin layer is connected to the glial cell body through cytoplasmic channels,

which contain cytoskeleton, vesicles and outposts of endoplasmic reticulum and Golgi

apparatus [3] and is considered relevant for the maintenance and turnover of myelin.

Schwann cell-derived myelin of the peripheral nervous system (PNS) differs from

oligodendrocyte-derived myelin of the central nervous system (CNS) regarding ontogenic

origin (subventricular zone versus neural crest), number of myelinated axonal segments per

glial cell (1:1 versus up to 60:1), and major proteins (the Ig-CAM myelin protein zero, (P0,

official gene name Mpz) versus the tetraspan proteolipid protein (PLP, Plp1)) (for review see

[4]).

Many aspects of myelin biogenesis, maintenance and metabolism, and of the function of

oligodendrocytes in preventing myelinated axons from degeneration [5, 6] are not well

understood at the molecular level. In this review, we focus on systematic analyses aimed at a

molecular understanding of CNS myelin. First we - briefly - consider systematic analyses at

the level of mRNA, miRNA and DNA, which have recently provided a wealth of information

regarding the mechanisms that regulate myelination and remyelination. Secondly, we review –

more extensively – current advancements in the analysis of the protein composition of myelin

itself. The addition of proteomic techniques to the methodological spectrum applied in myelin

research has accelerated our knowledge of which proteins are actually myelin constituents,

thereby providing a versatile basis for the systematic analysis of their functions in the

physiology and pathophysiology of myelinated fiber tracts. We have recently reviewed myelin

proteome analyses with a focus on technical [7] and disease-related [8] aspects. Here we

provide a meta-analysis of CNS myelin protein composition on the basis of unbiased protein

identification by mass spectrometry (MS) and the bioinformatic integration with other

systematic approaches such as mRNA microarray analysis.

Regulators of myelination identified by systematic mRNA, miRNA and DNA

analyses

Profiling of mRNA abundance during the differentiation of oligodendrocyte progenitor cells

(OPC) to post-mitotic, pre-myelinating oligodendrocytes (OL) and myelinating

oligodendrocytes (mOL), led to the identification of extensive changes affecting gene

clusters involved in the cell cycle, cell motility, adhesion, cytoskeletal remodeling, lipid

metabolism and ultimately myelination [9-12]. Filtering of such datasets allowed the

elucidation of functions as diverse as the regulation of OPC migration by semaphorins and

neuropilins [13], the transcriptional control of oligodendrocyte maturation and myelination

by ‘myelin gene regulatory factor‘ (Mrf, also termed gene model 98, Gm98) [14] and the

modulation of OPC differentiation and remyelination involving the nuclear ‘retinoid X receptor

gamma‘ (Rxrg) [15]. Additionally, novel proteins, such as an oligodendrocytic cell adhesion

molecule termed ‘proline-rich membrane protein‘ (PRMP/TMEM58, official gene name Shisa4)

[11] and ‘oligodendrocytic myelin paranodal and inner loop protein‘ (Opalin, also termed

transmembrane protein 10, TMEM10) [16, 17] have been identified.

Importantly, the expression of genes in oligodendrocytes is not only controlled by

transcriptional [18, 19], but also by post-transcriptional and post-translational modifications.

For example, oligodendroglial differentiation [20] coincides with alterations in the abundance

of regulatory RNAs [21-24] including miR-219, which represses proteins that both, promote

OPC proliferation and negatively regulate maturation. The regulation of oligodendrocyte

differentiation and myelination by epigenetic factors, including histone modifications, DNA

methylation and microRNAs, has been extensively reviewed [25-32], also in the context of

demyelinating disease [32-40]. Collectively, the studies discussed in these overview articles

founded the concept that the final step of oligodendroglial differentiation necessitates not

only transcriptional activation of myelin-related genes but also the concurrent de-repression

of various inhibitors of myelination [41, 42]. Taken together, systematic analyses at the level

of mRNA, miRNA and DNA have recently provided many important insights into regulatory

networks in oligodendrocytes, and more generally in their physiology and pathophysiology.

However, since not all mRNAs expressed in oligodendrocytes encode myelin proteins and not

all myelin proteins are signified by the same mRNA abundance profile, studies at the

nucleotide level are not well suited to systematically assess the protein content of myelin.

Towards the myelin proteome

In the early 1970s William Norton and Shirley Poduslo introduced a method to biochemically

enrich myelin from homogenized nervous tissue utilizing sucrose gradient centrifugation

[43], with myelin accumulating as low-density membranous material at the interface between

0.32 M and 0.85 M sucrose. As the method has proven to efficiently yield highly pure myelin,

only moderate modifications have been introduced since. These include the use of a Polytron

to substitute the Dounce homogenizer [44], a supplementary second density gradient

centrifugation that reduces axonal contaminations [45] and the addition of protease

inhibitors [46]. About the same time, the belief has formed that the number and variety of

myelin proteins is very low. This view emerged because upon one-dimensional gel-separation

of purified myelin and subsequent protein staining only very few bands were visible. These

are mainly constituted by PLP, myelin basic protein (Mbp) and 2’,3’-cyclic nucleotide

phosphodiesterase (Cnp1, previously termed ‘Wolfgram’s protein’) [47-51]. However, these

highly abundant proteins overshadowed low-abundance proteins that did not constitute

visible bands in early studies. Since then, the use of gradient-gels, two-dimensional gels, and

modified buffers and detergents has enhanced the migration of myelin into the gels and its

separation. Additionally, the sensitivity of modern staining procedures based on silver or

colloidal Coomassie is superior to the previously used Sudan Black and Fast Green. Together,

these improvements have allowed the visualization of many more bands (Fig. 1B), reflecting

that the protein composition of myelin is much more complex than previously anticipated.

To date, elaborated MS technology has turned out to be the most sensitive method to

systematically identify the protein constituents of myelin. While only one study of the PNS

myelin proteome is available so far [52], eight proteomic studies of CNS myelin have been

published, providing 11 datasets for myelin from mouse [7, 53-56], rat [57, 58] and human

[56, 59]. The approaches taken differ regarding details of sample preparation,

protein/peptide separation, MS instrumentation, and data analysis, including stringency

criteria for database search and acceptance of a protein hit. These differences

notwithstanding, we have compiled and compared these datasets ‘as published’ for the

present meta-analysis.

A compendium of mass spectrometrically identified myelin proteins

The integration of all myelin proteome datasets yielded a compendium of 1280 proteins,

among them 1261 for which it was possible to determine unique gene identifiers

(Supplemental Tab. S1). The number of proteins identified per approach markedly increased

in the more recent studies (Fig. 2A), reflecting advancements in proteomic methodology in

general and of MS instrumentation in particular. To measure the reproducibility of MS-based

myelin protein analyses we have determined which protein in any given dataset has

additionally been identified in at least one other approach. Reassuringly, more than half of all

proteins (52%) were present in at least two datasets (Fig. 2B), raising confidence in their

association with CNS myelin. The rate of reproduced identifications was almost 100% among

the earlier approaches (in which fewer than 150 proteins were identified) and well above

60% among the more recent approaches (with over 350 identified proteins) (Fig. 2C).

Importantly, the only exception is the single dataset in which - instead of conventionally

purified myelin - a remyelination-inhibiting subfraction was analyzed [58]. In that study, the

extensive prefractionation by ion exchange chromatography and multi-dimensional gel

electrophoresis led to a distinct sub-proteome, which is not directly comparable to that of

the complete myelin fraction. Together, this supports the view that the profile of myelin-

associated proteins as determined by sequential sucrose gradient centrifugation for myelin

purification and proteome analysis has been very robust across different laboratories,

irrespective of differences regarding the analyzed species, proteomic approach or MS

technology. We expect that further progress in MS performance will further increase the

coverage and the confidence level of the myelin proteome.

Interestingly, the only protein identified in all 11 approaches is not a ‘classical’ myelin protein

but a molecular chaperone termed heat shock 70kDa protein 8 (Hspa8; alternative name

constitutive heat shock protein, Hsc70). HSPA8 functions as a chaperone for MBP [60, 61]

and may thus be co-transported into myelin by molecular association. Five proteins were

each identified in 10 approaches, namely MBP, CNP, sirtuin 2 (SIRT2), septin 8 (SEPT8) and

dihydropyrimidinase-like 2 (DPYSL2, also termed collapsin response mediator, CRMP2) [53,

62-65]. Importantly, there was no simple correlation between the frequency of identification

and the abundance of a protein (Tab. 1). For example, the absence of transmembrane

proteins (such as PLP) among those most frequently identified was expected and is due to

the bias implemented by the use of conventional 2D gel electrophoresis in three approaches

[53-55]. This method comprises isoelectric focusing (IEF) as the first dimension of

separation, in which proteins migrate along an immobilized pH gradient until they reach their

isoelectric point (IP). However, hydrophobic membrane proteins tend to precipitate during

IEF, which has been attributed to the fact that proteins are least soluble at their IP while

potent ionic detergents such as SDS are not compatible with IEF. Together, this prevents the

transfer of membrane proteins to the second dimension SDS-PAGE [66], and thus their

identification.

Coinciding with the enhanced sensitivity of current MS instrumentation, the number of

identified contaminants from different cellular origins has increased. For example, proteomic

compendia of synaptic vesicles [67] or mammalian brain mitochondria [68] comprise

proteins derived from contaminating myelin, such as the myelin-specific proteins PLP, MBP,

myelin-associated glycoprotein (MAG) and myelin-oligodendrocyte basic protein (MOBP). Vice

versa, the most common contaminants of the myelin-enriched fraction are mitochondria and

synaptic vesicles, which can be attributed to similar floatation properties in sucrose or Percoll

gradients. Therefore it is not surprising that a considerable number of entries in the myelin

protein compendium may qualify as synaptic or mitochondrial according to prior knowledge.

However, information on subcellular localization, as assembled in large protein databases,

may be only partially true or even incorrect. For example, information for a particular protein

may have been assumed for an isoenzyme or another protein from the same biochemical

cascade without further validation. Moreover, many proteins supposed to be specific to

mitochondria actually do have a dual localization [68]. Thus, particular proteins may indeed

be present in both mitochondria and myelin, though antibody-based validation is lacking. We

also note that some proteins, such as neural cell adhesion molecule 1 (Ncam1), neurofascin

(Nfasc), and contactin 2 (Cntn2, also termed transient axonal glycoprotein, TAG-1) reside in

both, axonal membranes and (adaxonal) myelin [69-71]. In consequence, we have not

excluded proteins from the present compendium based on anticipated knowledge of

subcellular or cellular localization other than myelin.

The integration of the myelin proteome with other systematically gained information

circumvents some of the limitations of prior knowledge. For example, transcriptomic datasets

can clarify cellular origins and developmental mRNA abundance profiles (see below).

Bioinformatic approaches cannot only predict subcellular localizations, but also group

proteins according to structural criteria such as the presence of particular domains. For the

present compendium we have systematically predicted mitochondrial proteins utilizing Cello

(cello.life.nctu.edu.tw), TargetP (cbs.dtu.dk/services/TargetP) and Wolfpsort (wolfpsort.org)

and transmembrane proteins with the algorithms TMHMM2 v2.0

(cbs.dtu.dk/services/TMHMM), Phobius (phobius.sbc.su.se) and TMpred

(ch.embnet.org/software/TMPRED_form.html) (Supplemental Tab. S1). The numbers of

transmembrane and suspected mitochondrial proteins are indicated in Fig. 2A.

Cross-species comparison

The protein composition of myelin is considered highly similar among mammalian species [72,

73]. However, the cross-species comparison of MS-identified CNS myelin proteins of human

versus rodent origin (Fig. 3A) showed a surprisingly moderate overlap of only approximately

50%. Likely, the limited overlap reflects species-specific variations of amino acid sequences

affecting the proteolytic peptides rather than the existence of proteins exclusive to myelin of

one mammalian species. This view is supported by the finding that several proteins that upon

MS-analysis seemed specific to either human or rodent myelin were subsequently also

identified in the respective other species by immunoblot [56].

Comparison of CNS with PNS myelin

The proportion of proteins identified in both peripheral and central myelin is unexpectedly

high when the PNS myelin proteome [52] is compared with the present compendium (Fig.

3B). Proteins present in both types of myelin are likely relevant for functions or

morphological features that unify myelin in both the CNS and the PNS. For example, MBP,

which is considered relevant for the intracellular compaction of myelin membranes, is equally

abundant in both, CNS and PNS myelin. On the other hand, claudin 11 (Cldn11, also termed

oligodendrocyte specific protein, OSP) and periaxin (Prx) define molecular specializations of

the radial component particular to CNS myelin [74] and of the PNS-specific bands of Cajal

[75], respectively. Consequently, claudin 11 and periaxin were identified exclusively in CNS or

PNS myelin, respectively. This indicates that molecules specific to either type of myelin may

signify particular functions or morphological specializations. The relative abundance of

selected myelin proteins according to quantification by MS [7, 52] is compiled in Tab 1.

Comparison with transcriptomics data indicates that at least 90% of the

myelin-associated proteins are indeed robustly expressed in oligodendrocytes

The proteins identified in purified myelin by MS may include contaminants of a different

cellular origin that may have co-purified with myelin. As the individual validation of so many

proteins using antibody-based techniques is not feasible, we sought systematic information

regarding which proteins are actually expressed in oligodendrocytes. For this purpose we

have exploited a previously established transcriptional profile of oligodendrocytes, which

were isolated at distinct differentiation stages (PDGFRα+/MOG- OPC; GalC+ OL; MOG+/NG2-

mOL) by the team around John Cahoy and Ben Barres [9]. By filtering that dataset for the

proteins identified by MS in purified myelin, we have gained a bioinformatic comparison of the

myelin proteome and the oligodendrocyte transcriptome (Fig. 4, Supplemental Tab. S1). An

unambiguous correlation was possible for 1249 proteins and the corresponding mRNA.

For 79 proteins, no corresponding oligonucleotides were present on the arrays. For a further

106 proteins, corresponding mRNA was not detected in oligodendrocytes. This group may

include false-negatives for which the oligonucleotides on the arrays are not suitable, but also

proteins indeed not expressed in oligodendrocytes but co-purified with CNS myelin. The latter

category probably includes marker proteins of peripheral myelin such as P0 (Mpz), ‘peripheral

myelin protein 2‘ (Pmp2) [76] and ‘discs large homolog 1‘ (Dlg1) [77]. The identification of

these proteins in a very small subset of CNS myelin analyses may be attributed to myelin of

peripheral nerves inadvertently attached to the brains used as the starting material for

myelin purification.

Most importantly, robust mRNA abundance in oligodendrocytes at either differentiation stage

was found for at least 1064 (>90%) of the 1170 proteins identified in CNS myelin for which

such information was available. This suggests that contamination of the myelin-enriched brain

fraction by material from different cellular origins was fairly moderate.

Abundance profi les of mRNAs encoding CNS myelin proteins

To determine groups of myelin-associated genes signified by co-regulated expression, we

performed k-means cluster analysis for those 1064 mRNAs that were robustly detected in

oligodendrocytes. 631 mRNAs displayed significant abundance changes during

oligodendroglial differentiation (Fig. 4).

The majority of ‘classical’ myelin proteins clustered together with a significant mRNA-

abundance increase coinciding with the differentiation from OPCs to post-mitotic

oligodendrocytes (cluster ‘Early-UP’ in Fig. 4). These include Plp1 [4], Mbp [62], Cnp1 [64],

Sirt2 [53], Mag [78], Mobp [79], Cldn11 [74], Nfasc [70], myelin and lymphocyte protein

(Mal) [80], prominin 1 (Prom1) [81], the myelin-associated inhibitor reticulon 4 (Rtn4, Nogo)

[82], tubulin beta 4 (Tubb4) [83] and band 4.1 protein G (Epb4.1l2) [84]. A small cluster

(Up-DOWN in Fig. 4) comprised mRNAs with a temporarily increased abundance at the onset

of myelination such as the non-receptor tyrosine kinase fyn, which is involved in the

maturation of oligodendrocytes [85]. A third cluster of mRNAs that display a continuous

abundance increase during the differentiation from OPC to OL to mOL (cluster ‘Ascending’ in

Fig. 4) comprised the myelin marker carbonic anhydrase 2 (Car2) [86], Opalin [16, 17],

Cd82 [87], Rab3a [88], myelin oligodendrocyte glycoprotein (Mog) [89], integrin-associated

signal transducer (Cd47) [90], ermin (Ermn) [91], fatty acid synthase (Fasn) [92],

tetraspanin 2 (Tspan2) [93] and vesicle-associated membrane protein 3 (Vamp3) [94],

among many other proteins. It will be a task of future analyses to determine their role in

myelin biology.

Enhanced expression at the onset of myelination has often been considered circumstantial

evidence of a protein being a ‘myelin protein’. On the other hand, developmental repression

appears equally relevant. This is illustrated by the effect of polysialic acid (PSA), a

posttranslational modification of neural cell adhesion molecule (NCAM). Diminishment of

polysialylation coincides with the differentiation from OPC to mOL [69] and is a prerequisite

for efficient myelin biogenesis [95]. Thus, the early expression of both glial and axonal [96]

PSA-NCAM is inhibitory for premature myelination. Interestingly, the abundance of Ncam1

mRNA in oligodendrocytes is also repressed during the differentiation from OPC to mOL

(cluster ‘Descending’ in Fig. 4).

The 18kDa membrane-tetraspan peripheral myelin protein (PMP22) has long been assumed to

be absent from CNS myelin but was recently detected in purified CNS myelin by immunoblot

[97], though at a low level. Of note, identification of PMP22 by MS as in one approach [59] is

difficult by conventional proteomic approaches based on digestion with trypsin because its

cleavage sites are atypically distributed (leading to an unusually large 10 kDa fragment not

suitable for MS sequencing), and 65% of its amino acid residues constitute transmembrane

domains (leading to membrane-spanning peptides usually not detected by MS). Importantly,

by microarray analysis and in situ-hybridization, Pmp22 mRNA was detected in

oligodendrocytes [9, 21], in which its abundance decreases coinciding with the

differentiation from OPC to OL (‘Early-DOWN’ in Fig. 4), involving repression by miR-9 [21].

It is of note that developmental repression occurs frequently in oligodendrocytes: Among all

myelin-associated proteins, clusters with generally repressed abundance of the corresponding

mRNA (‘Early-DOWN’, ‘Descending’, ‘Late-DOWN’) comprise about as many genes as those

with increased mRNA abundance (‘Early-UP’, ‘Ascending’, ‘Late-UP’).

The abundance of 433 mRNAs encoding myelin-associated proteins, such as integrin beta 1

(Itgb1) [98], cell division cycle 42 (Cdc42), ras-related C3 botulinum toxin substrate 1

(Rac1) [99] as well as glyceraldehyde-3-phosphate dehydrogenase (Gapdh), was unchanged

during oligodendrocyte differentiation. Taken together, all clusters comprised previously

known myelin-related genes, supporting the notion that myelin proteins are not necessarily

signified by a particular developmental mRNA abundance profile.

Protein groups defined by homology or structural similarity

The systematic identification of protein groups with homology may be beneficial for

functional analysis. For example, by bioinformatic analysis of the compendium, those myelin-

associated proteins can be systematically identified that contain a FERM-domain - i.e. a

sequence motif with homology to four-point-one (4.1)-proteins, ezrin, radixin and moesin -

for molecular interactions (Epb41l1/4.1N, Epb41l2/4.1G, Epb41l3/4.1B, Ezr, Rdx, Msn, Tln1,

Tln2), or immunoglobulin-like domains that signify cell adhesion molecules (Ig-CAMs) (Alcam,

Bsg, Cadm2/NECL3, Cadm3/NECL1, Cadm4/NECL4, Cntn1, Cntn2/TAG1, Cntnap1/CASPR1,

Cntnap2/CASPR2, Dscaml1, Hepacam/GlialCAM, Igh, Igk, Igl, Igsf8, Itgb1, Itgb8, L1cam, Mag,

Mog, Mpzl1, Ncam1, Ncam2, Nrcam).

To further illustrate such systematic identification we have filtered tetraspan-transmembrane

proteins upon predicting the transmembrane domains for all proteins in the compendium

(Supplemental Tab. S1). Tetraspans are particularly abundant in myelin and have a proven

relevance for its biogenesis and pathology [100]. Tetraspan-encoding mRNAs with increased

abundance during maturation include Cd82, which restricts OPC migration [87] (Cluster

‘Ascending’ in Fig. 4), and those that determine the structure of myelin at the intraperiod

lines (Plp1) [4], radial component (Cldn11) [74] and paranodes (Mal) [80] (Cluster ‘Early-UP‘

in Fig. 4). The functions of other myelin tetraspans with ‘Ascending‘ mRNA profiles (in Fig.

4) may be related but remain to be established, including those of Tspan2, CKLF-like

MARVEL transmembrane domain containing 5 (Cmtm5), synaptogyrin 2 (Syngr2) and

transmembrane protein 205 (Tmem205). On the other hand, the repression of Pmp22 in

differentiating oligodendrocytes (see above) may be attributed to its capacity to repress

cellular growth and to form cellular junctions [101-104]. Similarly, the developmental

repression of synaptogyrin 3 (Syngr3, cluster ‘Early-DOWN‘), Cd151, glycoprotein M6A

(Gpm6a) (‘Descending‘) and Cd9 (‘Late-DOWN‘) may reflect functions of these tetraspans

not required during, or even inhibiting oligodendrocyte differentiation. Conversely, the

abundance of several tetraspan-encoding mRNAs (Pllp, Cd81, Connexin 43 (Gja1),

glycoprotein M6B (Gpm6b), secretory carrier membrane proteins 1 and 5 (Scamp1,

Scamp5)) is developmentally unchanged, suggesting continued requirement of the gene

product.

Notably, the membrane of vesicular exosomes derived from multivesicular bodies [105, 106]

comprises a particular abundance of tetraspans, in which they may facilitate fission and

fusion [107]. Oligodendrocyte-derived exosomes may be required to locally dispose off

superfluous membrane and to transfer material to axons [108] and microglia [109].

However, they also counteract the extension of oligodendroglial plasma membrane, at least in

vitro [110], possibly reflecting a function in the spatial segregation of myelin sheaths in vivo

related to that of Nogo (Rtn4) [82, 111]. Strikingly, there is a high overlap between the

tetraspans of exosomes and of myelin, including PLP, CD9, CD81, CD82 and CD63

(tetraspanin 30). It is attractive to speculate on commonalities of the two compartments

regarding biosynthesis and function. Together, the integration of proteomic and

transcriptomic datasets provides a background to study protein groups with structural and

functional similarity and co-regulated expression.

Heritable myelin-related diseases

Hereditary diseases affecting myelination or the physiology of the white matter

(leukodystrophies or leukoencephalopathies) vary considerably regarding the affected genes

and the pathophysiology. Importantly, only a subset of the causative genes encodes myelin

proteins [112-117]. For example mutations affecting the PLP1-gene, which encodes the

most abundant protein of CNS myelin, PLP, cause the hypomyelinating leukodystrophy

‘Pelizaus-Merzbacher disease‘ or the allelic ‘spastic paraplegia type 2‘ [118-120].

As oligodendrocytes, astrocytes, microglia and neurons intimately interact, the cellular

pathologies of leukodystrophies are very complex. Indeed, the identification of novel

leukodystrophy disease genes, which has accelerated in recent years [121-125], has

facilitated - often unexpected - insights into the biology and the interactions of glial cells

[126]. Importantly, many leukodystrophies are caused by mutations that do not affect

‘classical‘ myelin genes, or even genes considered to be expressed specifically in cells other

than oligodendrocytes. This is exemplified by ‘hypomyelination and congenital cataract‘

(HCC), which is caused by mutations affecting the FAM126A gene [127] encoding the

primarily neuronal protein hyccin [128], or by ‘hereditary diffuse leukoencephalopathy with

spheroids‘ (HDLS), which is caused by mutations affecting the gene encoding ‘colony

stimulating factor 1 receptor‘ (CSF1R) [125], considered exclusively expressed in microglia

[129]. However, such prior knowledge may infer an unjustified bias in the search for the

pathomechanism. For example, astrocytes are commonly considered the primary site of

pathology in ‘Alexander’s disease‘. Here, the white matter degeneration coincides with the

emergence of aggregates (termed Rosenthal fibers) in astrocytes. Rosenthal fibers comprise

the product of the causative gene, which encodes the intermediate filament glial fibrillary

acidic protein (Gfap) [130, 131]. However, whether astrocytic Rosenthal fibers indeed

contribute to the emergence of myelin abnormalities has not been satisfactorily shown.

Strikingly, GFAP is commonly thought to be exclusive to astrocytes, while our meta-analysis

emphasizes that GFAP was identified by MS in purified myelin and that the corresponding

mRNA was detected in oligodendrocytes (cluster ‘ASCENDING‘; Fig. 4). This suggests that

the expression of GFAP is less restricted than anticipated, and that it ought not be excluded

that oligodendrocytes and myelin are primary sites of pathology in Alexander’s disease.

In Table 2 we have compiled a list of genes that fulfill three criteria: (1) the protein was

identified by MS in myelin, (2) the mRNA was robustly detected in oligodendrocytes, and (3)

mutations affecting the corresponding human gene cause a disease that includes pathology

of myelin or the white matter, at least in a subset of patients. For example, one of the

causative genes for megalencephalic leukoencephalopathy with subcortical cysts (MLC), glial

cell adhesion molecule (GlialCAM, official gene name Hepacam; Cluster Down-UP in Fig. 4),

encodes an abundant CNS myelin protein [132] but is also expressed in astrocytes [124,

133]. In the latter, GlialCAM is involved in the intracellular trafficking of MLC1, a

transmembrane protein with distant homology to sodium channels, to its normal localization

at the junctions between astrocytes and neighboring astrocytes or the endothelial cells of

the blood-brain-barrier. As MLC1 is also a causative gene in MLC [134], a failure of GlialCAM-

dependent trafficking of MLC1 to astrocytic junctions is very likely disease relevant.

However, the emergence of myelin vacuoles in a subset of MLC patients may potentially be

attributed to the presence of GlialCAM in normal CNS myelin.

Taken together, oligodendrocytes and myelin may well contribute to the pathogenesis in

white matter diseases in which the affected genes are erroneously thought to be expressed

mainly or exclusively in other cells. However, proof for the involvement of particular cell

types in the pathogenesis of any leukodystrophy must come from the analysis of cell-type

specific mutant mice, which is also prerequisite for the development of rational therapy

concepts. We propose that the present compendium of myelin proteins also provides a useful

resource to identify causative genes in association studies in which only chromosomal

segments (comprising many genes) are currently known.

Tools and perspectives

Until recently, the constituents of myelin were mainly approached by single gene analysis.

However, with the advent of ‘omics’ techniques it became evident that all myelin proteins are

embedded in a context of molecular networks involving co-regulated expression and physical

protein-protein interactions. The present meta-analysis integrates systematic information

gained by proteomic analysis of normal CNS myelin in 11 published datasets and by

transcriptional profiling of differentiating oligodendrocytes upon immunopanning, i.e. cell

purification using antibodies directed against stage-specific surface antigens [9].

In an alternative approach to obtain samples for transcriptional profiling of distinct cell types,

BAC-transgenic mice were generated in which cell-type specific promoters drive the

expression of the ribosomal protein L10a with an EGFP-tag suited to affinity-purify labeled

polysomes for the subsequent analysis of the associated RNA (‘translating ribosome affinity

purification‘, TRAP) [135]. For the oligodendrocyte lineage, the Olig2 promoter (active from

OPCs to mOL) and the Cmtm5 promoter (active in mOL) were used [136]. Interestingly,

CMTM5 is a proven constituent of peripheral myelin [52], while antibody-based validation as

an oligodendroglial protein is yet lacking. However, its occurrence in the present compendium

(Cluster ‘Ascending‘ in Fig. 4) suggests that CMTM5 is a myelin protein also in the CNS.

Thus, the TRAP study has identified over 1000 probes representing hundreds of mRNAs with

a high probability of being translated in oligodendrocytes, which is also supported by the

considerable number of known myelin-related genes in the dataset (supplemental table S5 in

[136]. Considering that transgene expression under control of the Cmtm5 promoter was

comparatively weak, the future variation of TRAP utilizing a stronger oligodendrocyte-specific

promoter may allow the complementation of immunopanning for future transcriptional

profiling approaches in comparative analyses of mouse models of myelin-related diseases.

However, the application of either technique may remain limited, e.g. when oligodendroglial

surface antigens or the activity of driver-promoters are altered as part of the pathology.

Large-scale in situ-hybridization as supplied in the ‘Allen Brain Atlas‘ (mouse.brain-map.org)

and subsequent sorting of labeling patterns (Supplemental Table 11 in [137] by the time of

publication allowed the identification of 79 mRNAs with a high probability of oligodendrocyte-

enriched expression. Reassuringly, 37 (47%) among them are also represented in the current

myelin proteome compendium. Many of the remaining oligodendrocyte-enriched mRNAs

encode enzymes of the lipid metabolism (Abca2, Edg8, Fa2h, Fabp5, Hmgcs1, Lass2, Npc1,

Ugt8) and known oligodendroglial transcription factors (Olig1, Olig2, Mrf/Gm98, Sox10).

A more direct strategy to identify transcription factors in the oligodendrocyte lineage

involved in silico-screening [138] of a previously established transcription factor expression

atlas based on in situ-hybridization in the developing mouse CNS (‘Mahoney atlas‘) [139].

Out of 1445 transcription factors in the pictorial, 87 displayed an embryonic mRNA labeling

pattern compatible with expression in glial progenitors, and 20 displayed sustained glia-like

labeling at birth. Among those, 8 were already known to be involved in glial development

while 9 were newly identified as enriched in OPCs. One of them, the HMG-box transcription

factor 7-like 2 (Tcf7l2), was shown to be functionally involved in OPC maturation [138, 140,

141]. Together, the exploitation of pictorials allows the identification of molecules relevant

for oligodendrocytes, and thus is promising also beyond the application to transcription

factors. However, the interpretation of expression atlas data, their validation and their

integration with other systematic datasets [142-147] remains a challenge.

In conclusion, a wealth of systematically gained molecular information has recently emerged

for the normal development of oligodendrocytes and their accompanying non-myelinating

cells. As of today, the exploitation of these resources is still in its infancy, and the field is

confronted with the luxury problem to choose the most interesting candidate proteins for

individual functional analysis. We believe that the integration of systematic datasets - as

illustrated here - can facilitate the selection of proteins for in-depth follow-up studies. The

current technical limitations of systematic approaches, including non-represented genes and

unsuited probes (affecting microarrays and pictorials), non-specific antibodies

(immunohistochemistry) and proteins unsuited for MS sequencing upon trypsin digest

(proteomics), may well be overcome, e.g. by whole-transcriptome sequencing, more specific

antibodies and alternative proteases, respectively. Rather, the systematic application beyond

normal tissue is laborious and expensive and may thus remain limited at last. However,

disease-relevant insights into the pathophysiology of the white matter eventually require

comprehensive knowledge of the spatio-temporal expression of all mRNAs, regulatory RNAs

and proteins, not only in the normal brain but also in disease models and ultimately in

patients. While there are obvious limitations to the availability of human brain samples,

techniques for differential analyses of models of myelin disease have been established at the

proteomic [53, 148] and transcriptomic [14, 149, 150] level. It is encouraging that also the

application to complex traits in humans, such as multiple sclerosis [151-157] and psychiatric

diseases [158-162], has been initiated.

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Figure legends

Figure 1. Central nervous system myelin.

(A) The optic nerve of an adult, wild-type mouse was visualized by transmission electron

microscopy upon fixation by high-pressure freezing and freeze substitution. Several

myelinated axons are shown in cross-section. Note the periodic arrangement of myelin

membranes. Electron micrograph kindly provided by W. Möbius. (B) One-dimensional gel-

separation of CNS myelin. Myelin purified from wild-type mouse brains was separated by SDS-

PAGE in different buffer systems providing improved resolution either in the low (MES) or

high (MOPS) molecular weight range. Proteins were visualized with colloidal Coomassie

(Coom., 5 µg protein load) or silver staining (0.5 µg protein load). Bands are denoted, which

are constituted by known myelin proteins according to mass spectrometric identification.

MAG, myelin associated glycoprotein; TUBA, α-tubulin; CNP, 2’,3’-cyclic nucleotide

phosphodiesterase; SIRT2, sirtuin 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;

CLDN11, claudin 11/OSP; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein;

MBP, myelin basic protein; CKB, brain creatine kinase; CA2, carbonic anhydrase 2; MOBP,

myelin-associated oligodendrocytic basic protein. In bands marked with arrowheads, only

proteins not previously associated with myelin were identified.

Figure 2. Assembling a compendium of CNS myelin proteins.

(A) The number of proteins identified in different approaches to the CNS myelin proteome is

plotted. The total number of myelin-associated proteins is unknown. Transmembrane proteins

(black) were systematically predicted by TMHMM2, Phobius and TMpred software. Proteins

derived from mitochondria (which are diminished but not entirely lost during myelin

purification) were predicted by Cello and Wolfpsort software and according to the literature.

T [55]; V [54]; R [57]; W [53]; B [58]; J [7]; D [59]. I [56] provide datasets for mouse (I-

m) and human (I-h) myelin. The integration of all datasets (‘All’) yields a catalogue of 1261

proteins for which a unique gene identifier was available. (B) Single and multiple

identifications. For all proteins identified in CNS myelin it was plotted in how many

approaches they were identified. Note that fewer than half of the proteins (48%) were

identified in only one approach. (C) Cross-study reproducibility. For all approaches to the

CNS myelin proteome it was plotted which percentage of identified proteins were additionally

identified in at least one other approach. Note the high overall reproducibility. The seemingly

low reproducibility of the dataset B [58] is due to the subfractionation of myelin by ion

exchange chromatography (IEX) (see text for details).

Figure 3. Venn diagram comparing the number of proteins identified in human versus rodent

CNS myelin (A), and in CNS versus PNS myelin (B) according to [52].

Figure 4. Profi les of myelin-associated mRNAs in oligodendrocytes.

(A) The mRNA abundance profiles in oligodendrocyte progenitor cells (OPC), pre-myelinating,

post-mitotic oligodendrocytes (OL) and myelinating oligodendrocytes (mOL), as determined

by [9], were filtered for the proteins identified by MS in purified myelin. Upon k-means

clustering, the normalized mRNA-abundance profiles were plotted with regard to the

differentiation stage. Genes with significant mRNA-abundance changes were categorized in

eight clusters. Known myelin-related genes are in bold, and genes encoding proteins probably

derived from mitochondrial, blood or nuclear contamination are in gray. mRNAs in the clusters

‘UP’ or ‘ASCENDING’ display significantly increased abundance during oligodendrocyte

differentiation while mRNAs in the clusters ‘DOWN’ or ‘DESCENDING’ are significantly

suppressed during development. (B) The numbers of mRNAs per cluster are given.

Supplemental Table S1. Compendium of proteins identified by mass

spectrometry in CNS myelin.

Out of the 1280 proteins listed, 1261 proteins could be assigned to a unique gene identifier,

of which 1249 could be correlated with the oligodendroglial transcriptome as established by

[9] (last column). The proteins are classified into three groups: known myelin proteins,

proteins identified by MS in myelin, and proteins presumably derived from mitochondria that

contaminate the myelin-enriched fraction. Identification of a protein in one of the 11 available

proteomic datasets (see main text for references) is indicated by "1". Potential mitochondrial

localization was classified by prior knowledge (MitoCarta) and prediction of subcellular

localization according to three algorithms (TargetP, Cello, Wolfpsort). The number of

transmembrane domains was predicted by three algorithms (TMpred, TMHMM, Phobius). If

available, correlation with a large-scale in situ-hybridization dataset (‘Allen Brain Atlas‘) is

provided (OL, oligodendrocyte; AS, astrocyte; NE, neuron) according to supplemental table

11 in [137].

Table 1. Relative abundance of major myelin proteins by mass spectrometric quantification Protein CNS myelin (%) PNS myelin (%) PLP 17 0.2 MBP 8 8 CNP 4 0.5 MOG 1 nd MAG 1 0.3 SIRT2 1 nd OSP 1 nd P0 nd 21 Periaxin nd 16 FASN nd 1 4.1G nd 1 Others 67 52 Proteins associated with purified CNS or PNS myelin were identified and quantified by LC-MSE [7, 52]. Selected myelin proteins are sorted by their relative abundance in CNS myelin. nd, not detected.

Table 2. Comparison of proteins identified in CNS myelin and disease genes associated with pathology of myelin or the white matter Gene Protein name mRNA profile OMIM Disease Remarks AHCY S-Adenosylhomocysteine Hydrolase Unchanged *180960

#613752 Hypermethioninemia Slow myelination, white

matter atrophy CTSD Cathepsin D Unchanged *116840

#610127 Ceroid Lipofuscinosis White matter

abnormalities GFAP Glial Fibrillary Acidic Protein Ascending *137780

#203450 Alexander Disease Leukodystrophy

GLUL Glutamate-Ammonia Ligase Late-UP *138290 #610015

Glutamine Deficiency Hypomyelination

HEPACAM Glial Cell Adhesion Molecule Down-UP *614133 #613926 #613925

Megalencephalic Leukoencephalopathy with Subcortical Cysts 2

Leukoencephalopathy

L1CAM L1 Cell Adhesion Molecule Descending *308840 #304100 #303350

Spastic Paraplegia type 1; Agenesis of the Corpus Callosum; MASA

White matter abnormalities

MBP Myelin Basic Protein Early-UP *159430 #601808

18q Deletion Syndrome Dysmyelination; Deleted chromosome segment includes MBP

MOG Myelin-Oligodendrocyte Glycoprotein Ascending *159465 #614250

Narcolepsy 7 Unknown pathogenesis

NPC1 Niemann-Pick C1 protein Unchanged *607623 #257220

Niemann-Pick Disease type C1

Impaired myelination

PHGDH Phosphoglycerate Dehydrogenase Late-UP *606879 #601815

PHGDH deficiency Dysmyelination

PLP1 Myelin Proteolipid Protein Early-UP *300401 #312080 #312920

Pelizaeus-Merzbacher Disease; Spastic Paraplegia type 2

Hypomyelinating leukodystrophy

PSAP Prosaposin Unchanged *176801 #249900 #611722

Metachromatic Leukodystrophy; Krabbe Disease

Leukodystrophy

PSAT1 Phosphoserine Aminotransferase Early-UP *610936 #610992

Phosphoserine amino-transferase deficiency

Poor white matter development

SLC12A6 Na/Cl-Cotransporter 3 Down-UP *604878 #218000

Agenesis of the Corpus Callosum

White matter abnormalities

SOD1 Superoxide Dismutase, Cytosolic Unchanged *147450 #105400

Amyotrophic lateral sclerosis Demyelination

Peroxisomal or dual localization ALDH3A2 Aldehyde Dehydrogenase 3a2 Ascending *609523

#270200 Sjogren-Larsson Syndrome Spasticity, paraplegia

HSD17B4

17-Beta-Hydroxysteroid Dehydrogenase IV

Unchanged *601860 #261515

Bifunctional protein deficiency

Dysmyelination

Mitochondrial or dual localization ETFA Electron Transfer Flavoprotein a Late-DOWN *608053

# 231680 Glutaric acidemia IIA Multisystemic, with

leukoencephalopathy HSPD1 Heat Shock 60kDa Protein 1 Late-DOWN *118190

#612233 Hypomyelinating Leukodystrophy 4

Leukodystrophy

NDUFV1 NADH-Ubiquinone Oxidoreductase Flavoprotein 1

Unchanged *161015 #203450 # 256000

Alexander Disease; Leigh syndrome

Demyelination

SLC25A12 Aspartate-Glutamate Carrier Aralar Unchanged *603667 #612949

Global cerebral hypomyelination

Hypomyelination

Probably mitochondrial localization AUH Au-specific RNA-binding Protein Ascending *600529

#250950 3-Methylglutaconic Aciduria White matter lesions

Proteins are listed that fulfill three criteria: (1) the protein was identified by MS in purified CNS myelin, (2) the corresponding mRNA was detected in oligodendrocytes and (3) mutations affecting the corresponding gene are associated with diseases that reportedly can include pathology of myelin or the white matter. Localization to mitochondria, which partly co-purify with myelin, was designated according to software-based prediction and a brain mitochondrial proteome study [68]. Note that mutations affecting mitochondrial proteins may infer myelin-related pathology. For proteins with additional expression in astrocytes, microglia, or neurons it is presently unknown whether loss/gain of function in oligodendrocytes is indeed causative of the disease. The mRNA abundance profile according to k-means cluster analysis (Fig. 4) is given.

Figure 1. Central nervous system myelin. (A) The optic nerve of an adult, wild-type mouse was visualized by transmission electron microscopy upon �xation by high-pressure freezing and freeze substitution. Several myelinated axons are shown in cross-section. Note the periodic arrangement of myelin membranes. Electron micrograph kindly provided by W. Möbius. (B) One-dimensional gel-separation of CNS myelin. Myelin puri�ed from wild-type mouse brains was separated by SDS-PAGE in di�erent bu�er systems providing improved resolution either in the low (MES) or high (MOPS) molecular weight range. Proteins were visualized with colloidal Coomassie (Coom., 5 µg protein load) or silver staining (0.5 µg protein load). Bands are denoted, which are constituted by known myelin proteins according to mass spectrometric identi�cation. MAG, myelin associated glycoprotein; TUBA, α-tubulin; CNP, 2’,3’-cyclic nucleotide phosphodiesterase; SIRT2, sirtuin 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CLDN11, claudin 11/OSP; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein; MBP, myelin basic protein; CKB, brain creatine kinase; CA2, carbonic anhydrase 2; MOBP, myelin-associated oligodendrocytic basic protein. In bands marked with arrowheads, only proteins not previously associated with myelin were identi�ed.

Figure 2. Assembling a compendium of CNS myelin proteins. (A) The number of proteins identi�ed in di�erent approaches to the CNS myelin proteome is plotted. The total number of myelin-associated proteins is unknown. Transmembrane proteins (black) were systematically predicted by TMHMM2, Phobius and TMpred software. Proteins derived from mitochondria (which are diminished but not entirely lost during myelin puri�cation) were predicted by Cello and Wolfpsort software and according to the literature. T [55]; V [54]; R [57]; W [53]; B [58]; J [7]; D [59]. I [56] provide datasets for mouse (I-m) and human (I-h) myelin. The integration of all datasets (‘All’) yields a catalogue of 1261 proteins for which a unique gene identi�er was available. (B) Single and multiple identi�cations. For all proteins identi�ed in CNS myelin it was plotted in how many approaches they were identi�ed. Note that fewer than half of the proteins (48%) were identi�ed in only one approach. (C) Cross-study reproducibility. For all approaches to the CNS myelin proteome it was plotted which percentage of identi�ed proteins were additionally identi�ed in at least one other approach. Note the high overall reproducibility. The seemingly low reproducibility of the dataset B [58] is due to the subfractionation of myelin by ion exchange chromatography (IEX) (see text for details).

Figure 3. Venn diagram comparing the number of proteins identi�ed in human versus rodent CNS myelin (A), and in CNS versus PNS myelin (B) according to [52].

Figure 4. Pro�les of myelin-associated mRNAs in oligodendrocytes.(A) The mRNA abundance pro�les in oligodendrocyte progenitor cells (OPC), pre-myelinating, post-mitotic oligodendrocytes (OL) and myelinating oligodendrocytes (mOL), as determined by [9], were �ltered for the proteins identi�ed by MS in puri�ed myelin. Upon k-means clustering, the normalized mRNA-abundance pro�les were plotted with regard to the di�erentiation stage. Genes with signi�cant mRNA-abundance changes were categorized in eight clusters. Known myelin-related genes are in bold, and genes encoding proteins probably derived from mitochondrial, blood or nuclear contamination are in gray. mRNAs in the clusters ‘UP’ or ‘ASCENDING’ display signi�cantly increased abundance during oligodendrocyte di�erentiation while mRNAs in the clusters ‘DOWN’ or ‘DESCENDING’ are signi�cantly suppressed during development. (B) The numbers of mRNAs per cluster are given.