Received: 4 September 2017 | Revised: 9 November 2017 | Accepted: 17 November 2017 DOI: 10.1002/glia.23274 RESEARCH ARTICLE Myelin extracellular leaflet compaction requires apolipoprotein D membrane management to optimize lysosomal-dependent recycling and glycocalyx removal Nadia García-Mateo1 | Raquel Pascua-Maestro1 | Alberto Perez-Castellanos 1 | Concepcion Lillo2 | Diego Sanchez1 | Maria D. Ganfornina1 1Instituto de Biología y Genetica Molecular- Departamento de Bioquímica y Biología Abstract Molecular y Fisiología, Universidad de To compact the extracellular sides of myelin, an important transition must take place: from mem- Valladolid-CSIC, Valladolid, Spain brane sliding, while building the wraps, to membrane adhesion and water exclusion. Removal of the 2Instituto de Neurociencias de Castilla y negatively charged glycocalyx becomes the limiting factor in such transition. What is required to ini- Leon, IBSAL, Universidad de Salamanca, tiate this membrane-zipping process? Knocking-out the Lipocalin Apolipoprotein D (ApoD), essential Salamanca, Spain for lysosomal functional integrity in glial cells, results in a specific defect in myelin extracellular leaflet Correspondence compaction in peripheral and central nervous system, which results in reduced conduction velocity Maria D Ganfornina, Instituto de Biología y and suboptimal behavioral outputs: motor learning is compromised. Myelination initiation, growth, Genetica Molecular, c/Sanz y Fores 3, intracellular leaflet compaction, myelin thickness or internodal length remain unaltered. Lack of Universidad de Valladolid-CSIC, 47003 Valladolid, Spain. ApoD specifically modifies Plp and P0 protein expression, but not Mbp or Mag. Late in myelin matu- E-mail: [email protected] ration period, ApoD affects lipogenic and growth-related, but not stress-responsive, signaling pathways. Without ApoD, the sialylated glycocalyx is maintained and ganglioside content remains Funding information high. In peripheral nervous system, Neu3 membrane sialidase and lysosomal Neu1 are coordinately Junta de Castilla y Leon (JCyL), Grant Number: VA180A11-2, MICINN-MINECO, expressed with ApoD in subsets of Schwann cells. ApoD-KO myelin becomes depleted of Neu3 and BFU2011-23978, and BFU2015-68149-R; enriched in Fyn, a kinase with pivotal roles in transducing axon-derived signals into myelin proper- European Regional Development fund; ties. In the absence of ApoD, partial permeabilization of lysosomes alters Neu1 location as well. JAEPre – CSIC fellowship; JCyL fellowship, Exogenous ApoD rescues ApoD-KO hypersialylated glycocalyx in astrocytes, demonstrating that Grant Number: call#EDU/1883/2013; European Social Fund; Operational Pro- ApoD is necessary and sufficient to control glycocalyx composition in glial cells. By ensuring lysoso- gramme for Castilla y Leon; Consejería de mal functional integrity and adequate subcellular location of effector and regulatory proteins, ApoD Educacion (JCyL) guarantees the glycolipid recycling and glycocalyx removal required to complete myelin compaction. KEYWORDS extracellular leaflet, gangliosides, lysosome, motor learning, myelin compaction 1 | INTRODUCTION followed by the construction of a few turns of incipient myelin. Signal- ing continues through the process of growth and maturation of myelin Myelinating glial cells have the important task of building and maintain- (reviewed by Baron & Hoekstra, 2010; Jacob, Lebrun-Julien, & Suter, ing large expansions of their membranes that enwrap axons and 2011; Pereira, Lebrun-Julien, & Suter, 2012; Quarles, Macklin, & Mor- provide the basis for saltatory nerve impulse propagation, thus condi- ell, 2006; Simons & Trotter, 2007; Snaidero & Simons, 2017; White & tioning the function of neural circuits. Complex glia-axon communica- Kramer-Albers, 2014). tion initiates the first contact with axons and induces glial cell polarity, Myelin maturation requires the exclusion of water-rich media from large compact myelin domains (Bakhti, Aggarwal, & Simons, 2014), Diego Sanchez and Maria D. Ganfornina contributed equally to this work. which results in intimate membrane appositions recognized by electron ....................................................................................................................................................................................... This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. VC 2017 The Authors GLIA Published by Wiley Periodicals, Inc. Glia.2017;1–18. wileyonlinelibrary.com/journal/glia | 1 2 | GARCIA-MATEO ET AL. microscopy as the major dense line (MDL, condensation of the intracel- diseases known so far, independently of the gene affected, are associ- lular space) and intra-period line (IPL, condensation of the extracellular ated to leukodystrophy or myelination problems (Nave & Werner, space). The complex process of myelination is a robust one, with many 2014; Renaud, 2012), and lysosomal-dependent membrane recycling regulatory “checkpoints” and redundant systems to safeguard the final has important implications for the correct location of Plp in CNS com- product: a healthy and properly myelinated axon. Knocking-out impor- pact myelin domains (Baron & Hoekstra, 2010; Trajkovic et al., 2006). tant structural or regulatory players often have less-than-expected We have recently discovered that the Lipocalin Apolipoprotein D effects. For example, functional myelination still occurs in specific brain (ApoD), already known to be required for adequate myelin manage- areas in the absence of Fyn, a kinase of the SLK family with pivotal ment after PNS injury (Ganfornina et al., 2010; Garcia-Mateo et al., roles in transducing and integrating axon-derived signals (reviewed by 2014), is targeted to lysosomes in astrocytes and neurons, and is Kramer-Albers & White, 2011; White & Kramer-Albers, 2014). required for lysosomal membrane stability and pH homeostasis (Pas- Myelin compaction of the already formed myelin layers can be dis- cua-Maestro, Diez-Hermano, Lillo, Ganfornina, & Sanchez, 2017). The sociated in two phases: intracellular and extracellular leaflet compaction. pH distribution of the subset of ApoD-positive lysosomes is in the While the former is well understood thanks to many studies on the role range of optimal pH for neuraminidases, also coincident with that of of myelin basic protein (Mbp) in myelin assembly (at cellular, biochemical secretory lysosomes. A lysosomal population with these properties and biophysical levels; reviewed by Aggarwal, Yurlova, & Simons, 2011; would be the perfect candidate to manage glycocalyx remodeling and Bakhti et al., 2014), the compaction of myelin extracellular sides remains myelin membrane recycling. This unexpected relationship of ApoD elusive. Experimental manipulations of key candidate proteins (protein with such a subset of lysosomes, and the evidences that myelin built in zero, P0, in peripheral nervous system [PNS] and proteolipid protein, Plp, an ApoD-KO background must have different properties altering mye- in central nervous system [CNS]), thought to mediate the required adhe- lin recognition and digestion by phagocytic cells (Garcia-Mateo et al., sion between extracellular leaflets, either produced major alterations in 2014; Pascua-Maestro et al., 2017), led us to study the role of ApoD in several myelination steps, affecting both intracellular and extracellular the development and maturation of myelin. leaflet compaction (Giese, Martini, Lemke, Soriano, & Schachner, 1992), By analyzing myelin formation and maintenance from postnatal or yielded less effects than expected on myelin compaction. Lack of Plp day 3 (P3) to aged mice (P630) we have found that lack of ApoD spe- does not prevent the formation of compact myelin (Klugmann et al., cifically alters the process of myelin extracellular leaflet compaction in 1997), and compact myelin regions are also found in P0-KO nerves both CNS and PNS without major defects in intracellular leaflet com- coexisting with severe defects in myelin sheaths (Giese et al., 1992), paction or other myelin properties. The functional consequences of this revealing again the robustness of the process. On the other hand, experi- specific alteration of the terminal phase of myelin maturation are mental manipulation of the glycocalyx of oligodendrocytes in culture explored, as well as the signaling pathways altered throughout myelina- modifies the adhesion of myelin particles (Bakhti et al., 2013). Currently, tion. We conclude that ApoD ultimately controls the removal of the the working hypothesis in the field is that removal of the hydrophilic gly- sialic-rich hydrophilic glycocalyx, by maintaining functional integrity of cocalyx is required, as a limiting factor, to allow for protein-protein inter- lysosomes. A detailed analysis of the mechanism in the PNS reveals actions between apposing myelin membranes (Bakhti et al., 2014; Bakhti that the proper localization (and hence activity) of both lysosomal et al., 2013). Within this paradigm, the role of P0 or Plp proteins is to Neu1 and plasma membrane Neu3, as well as of the membrane-bound maintain the “membrane zip” locked, not to initiate the “zipping” process Fyn kinase, depend on ApoD. Finally, we demonstrate that itself. However, no single mutant so far has been described that specifi- lysosomally-located ApoD