GLIA 58:1949–1960 (2010) A Proteome Map of Axoglial Specializations Isolated and Purified from Human Central Nervous System AJIT S. DHAUNCHAK,1* JEFFREY K. HUANG,1,2 OMAR DE FARIA JUNIOR,1 ALEJANDRO D. ROTH,1,3 1 1 1 1 LILIANA PEDRAZA, JACK P. ANTEL, AMIT BAR-OR, AND DAVID R. COLMAN * 1The Montreal Neurological Institute (MNI) and Hospital, McGill University and the McGill University Health Centre, Department of Neurology and Neurosurgery, Program in NeuroEngineering of McGill University, 3801 University Avenue, Montreal H3A2B4, Canada 2Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom 3Department of Biology, University of Chile, Santiago, Chile KEY WORDS progressively diminishing the ability to perform even myelin; node of Ranvier; myelin inhibition; multiple sclero- the simplest motor tasks. Several animal models, either sis; white matter disorder genetically-engineered or carrying spontaneous muta- tions, mimic human white matter disorders. These con- ditions have shed light on the underlying pathology of ABSTRACT several leukodystrophies, including Pelizeaus-Merz- Compact myelin, the paranode, and the juxtaparanode are discrete domains that are formed on myelinated axons. In bacher disease (PMD), X-linked Adrenoleukodystrophy humans, neurological disorders associated with loss of (ALD), 18q Syndrome (shiverer mice), Metachromatic myelin, including Multiple Sclerosis, often also result in Leukodystrophy (MLD). However, the underlying causes disassembly of the node of Ranvier. Despite the impor- of demyelinating (e.g., multiple sclerosis) versus dysmye- tance of these domains in the proper functioning of the linating ailments (e.g., leukodystrophies) remain poorly CNS, their molecular composition and assembly mecha- understood. In addition, why mutations in certain nism remains largely unknown. We therefore performed a housekeeping genes like eukaryotic initiation factor 2 large-scale proteomics MudPIT screen for the identification family proteins (eIF2B a-to-gamma) or genes associated of proteins in human myelin and axogliasomal fractions. with peroxisomal disorders result in white matter loss is We identified over 1,000 proteins in these fractions. Since still unclear. A better understanding of the underlying even minor perturbations in neuron-glial interactions can uncouple the glial support of axons, the proteome map mechanisms of disease requires generation of the pro- presented here can be used as a reference library for teome and transcriptome map of human OLs and mye- ‘‘myelin health’’ and disease states, including white matter lin, as has been attempted for rodents (Dugas et al., disorders such as leukodystrophies and multiple sclero- 2006; Roth et al., 2006; Taylor and Pfeiffer, 2003; Taylor sis. VC 2010 Wiley-Liss, Inc. et al., 2004; Vanrobaeys et al., 2005; Werner et al., 2007; Yamaguchi et al., 2008). Accordingly, we modified the myelin-isolation protocol and purified myelin and axo- INTRODUCTION gliasomes from freshly dissected human white matter tissue. The axoglial structures are remarkably stable Myelin ensheathment by oligodendrocytes (OL) in the and retain junctional morphology upon extraction with human central nervous system (CNS) ensures rapid high concentration detergent. We performed a large- impulse conduction. In addition to insulation, myelin scale nongel-based proteome screen on the biochemi- has also been shown to trigger domain formation on the cally-isolated fractions. We identified over 750 proteins axon (Pedraza et al., 2001; Poliak and Peles, 2003). My- in the human myelin fraction and over 450 in the axo- elinated axonal segments (internodes) are flanked by short unmyelinated segments (nodes of Ranvier). Volt- Additional Supporting Information may be found in the online version of this age-gated sodium channels are clustered at these nodes, article. limiting depolarization to this sharply demarcated zone; Grant sponsors: Rio Tinto Alcan, The Molson Foundation, The Myelin Repair Foundation, Center of Excellence Award [(2007) Government of Canada], MS Soci- as a result, action potentials ‘‘jump’’ from one node to ety of Canada Postdoctoral Fellowship. the next. The region immediately flanking the node is *Correspondence to: Ajit S. Dhaunchak, The Montreal Neurological Institute termed the paranode and the outermost domain is called (MNI) and Hospital, McGill University and the McGill University Health Centre, Department of Neurology and Neurosurgery, Program in NeuroEngineer- the juxta-paranode. For simplicity, we have termed the ing of McGill University, 3801 University Avenue, Montreal H3A2B4, Canada. axonal specializations together with the overlying and E-mail: [email protected] and David R. Colman, The Montreal Neurolog- ical Institute (MNI) and Hospital, McGill University and the McGill University attached glial paranodal loops as the ‘‘axoglial appara- Health Centre, Department of Neurology and Neurosurgery, Program in Neuro- tus’’ (Huang et al., 2005; Pedraza et al., 2001). How Engineering of McGill University, 3801 University Avenue, Montreal H3A2B4, Canada. E-mail: [email protected] these microdomains are assembled is a major focus of Received 26 March 2010; Accepted 22 July 2010 research efforts. DOI 10.1002/glia.21064 Genetic perturbations resulting in white matter Published online 9 September 2010 in Wiley Online Library (wileyonlinelibrary. abnormalities are eventually lethal in humans, often com) VC 2010 Wiley-Liss, Inc. 1950 DHAUNCHAK ET AL. glial fraction. When comparing the myelin proteome 4°C, the 0.32/1.0 M interface (crude myelin membranes), library to that of the axoglial compartment, we found and 1.0/1.25 M interface (axogliasomes) were collected, overlapping but distinct proteomes. We identified pro- diluted 1:1 with ice cold H2O and pelleted at 100,000g teins of diverse classes including metabolic, biosynthesis, for 1 h to concentrate the membranes. The axogliasomes and degradation machineries. The proteome map pre- and myelin fractions were hypoosmotically shocked by sented here is much larger than previously determined homogenization in 20 vol. of ice cold H2O, containing 20 in murine counterparts and we report over 400 proteins mM Tris pH 7.5 and 0.1 mM PMSF, incubated for 30 that were not known to be components of myelin. We also min on ice and rehomogenized. The shocked membranes present a proteome map of the axoglial fraction with over were pelleted at 40,000g for 20 min, resuspended in 0.32 400 proteins that were not described previously (Huang M sucrose and refloated to respective interfaces by ultra- et al., 2005). The proteome map presented here may centrifugation at 100,000g for 3 h. The collected mem- include novel autoantigens in multiple sclerosis, disease- branes were shocked once again before pelleting, and associated proteins in the discrete OL domains, and novel recovered for subsequent experiments. molecules inhibiting regeneration in the CNS. The human proteome map presented here thus can be used as Immunoblotting an atlas for myelin health and disease. Equal amounts of protein resolved on denaturing SDS-gel were transferred to PVDF membranes (Milli- MATERIALS AND METHODS pore). The membranes were rinsed briefly in TBST (25 Antibodies and Reagents mM Tris pH 7.4, 27 mM KCl, 137 mM NaCl, 0.1% Tween 20) and blocked for at least 1 h at room tempera- Monoclonal mouse obtained from following sources: ture in blocking buffer (5% nonfat dry milk in TBST). anti-PSD-95 (K28/43) and anti-panNeurofascin (A12/18) Primary antibody diluted in blocking buffer was applied from Neuromab, anti-Actin (A1978 Sigma), anti-Gfap for at least 1 h at room temperature (or overnight at (G-9269 Sigma), anti-Gapdh (G8795 Sigma), anti-Tubu- 4°C). After four washes in TBS-T, HRP-conjugated sec- lin (AA12.1 Hybridoma Bank, Iowa), claudin-11/OSP (a ondary antibodies were applied for 45 min, washed four gift of Dr. Alexander Gow, Wayne State University, times in TBS-T and developed using Enhanced Chemilu- Detroit, MI), anti-sodium channel (K58/35 Sigma), anti- minescence Detection kit (PerkinElmer). PLP clone 3F4 (Dhaunchak and Nave, 2007). Polyclonal rabbit were anti-Caspr (Tait et al., 2000), anti-Calnexin (3811-100 Biovision), anti-CNP (Bernier et al., 1987), Immunohistochemistry anti-OMgp (H-222 SantaCruz), anti-14-3-3 (K-19 Santa- Cruz), anti-MAG (Salzer et al., 1987), anti-MBP CNS tissues were obtained from postnatal day 18 rat spi- (Pedraza et al., 1997), Adam23 (a gift of Dr. Charles nal cords or brains after intercardial perfusion with 4% ffrench-Constant, Cambridge University, Cambridge, PFA, cryo-protected through a 12, 15, and 20% sucrose se- UK), Crmp-2 (a gift of Dr. Jerome Honnorat, INSERM riesandembeddedbyrapidfreezinginTissueTekOCTfor U433, France), plasmolipin (a gift of Dr. Victor Sapirst- cryosectioning. For sodium channel staining, the rats were ein, The Nathan Klein Institute, Orangeburg, NY), All perfused transcardially via 4% paraformaldehyde filled 30- chemicals and reagents used were from Sigma. cm3 syringes for no more than 5 min, and dissected tissues were postfixed for another 5–10 min before cryoprotection. Longitudinal and transverse sections (15 lm) were gener- Isolation and Purification of Myelin and Axoglial atedandplacedonSuperfrost/Plusglassslides(Fisher),and Apparatus Fractions stored at 280°C until use. PNS tissues were obtained from P18 rat sciatic nerves, fixed for 15 min in 4% paraformald- We obtained myelin from human tissue excised from hyde, transferred to 1X PBS, cut longitudinally, transferred