ORIGINAL RESEARCH published: 12 February 2019 doi: 10.3389/fncel.2019.00040 Cholesterol Depletion Regulates Axonal Growth and Enhances Central and Peripheral Nerve Regeneration Cristina Roselló-Busquets 1,2† , Natalia de la Oliva 2,3† , Ramón Martínez-Mármol 1,2†‡ , Marc Hernaiz-Llorens 1,2 , Marta Pascual 1,2,4 , Ashraf Muhaisen 1,2,4 , Xavier Navarro 2,3 , Jaume del Valle 2,3* and Eduardo Soriano 1,2,4,5* 1 Department of Cell Biology, Physiology and Immunology, Faculty of Biology, Institute of Neurosciences, University of Barcelona, Barcelona, Spain, 2 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain, 3 Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences, Universitat Autònoma de Barcelona, Bellaterra, Spain, 4 Vall d’Hebron Research Institute (VHIR), Barcelona, Spain, 5 ICREA Academia, Barcelona, Spain Axonal growth during normal development and axonal regeneration rely on the action of many receptor signaling systems and complexes, most of them located in Edited by: specialized raft membrane microdomains with a precise lipid composition. Cholesterol Stefania Raimondo, University of Turin, Italy is a component of membrane rafts and the integrity of these structures depends on the Reviewed by: concentrations present of this compound. Here we explored the effect of cholesterol Mehmet Emin Onger, depletion in both developing neurons and regenerating axons. First, we show that Ondokuz Mayıs University, Turkey Petr Dubový, cholesterol depletion in vitro in developing neurons from the central and peripheral Masaryk University, Czechia nervous systems increases the size of growth cones, the density of filopodium-like *Correspondence: structures and the number of neurite branching points. Next, we demonstrate that Jaume del Valle cholesterol depletion enhances axonal regeneration after axotomy in vitro both in a jaume.delvalle@gmail.com Eduardo Soriano microfluidic system using dissociated hippocampal neurons and in a slice-coculture esoriano@ub.edu organotypic model of axotomy and regeneration. Finally, using axotomy experiments in † These authors have contributed the sciatic nerve, we also show that cholesterol depletion favors axonal regeneration equally to this work ‡ Present address: in vivo. Importantly, the enhanced regeneration observed in peripheral axons also Ramón Martínez-Mármol, correlated with earlier electrophysiological responses, thereby indicating functional Clem Jones Centre for Ageing recovery following the regeneration. Taken together, our results suggest that cholesterol Dementia Research, Queensland depletion per se is able to promote axonal growth in developing axons and to Brain Institute, The University of Queensland, Brisbane, increase axonal regeneration in vitro and in vivo both in the central and peripheral QLD, Australia nervous systems. Received: 05 October 2018 Keywords: regeneration, cholesterol, lipid rafts, axon growth, growth cone, filopodia Accepted: 25 January 2019 Published: 12 February 2019 Citation: INTRODUCTION Roselló-Busquets C, de la Oliva N, Martínez-Mármol R, Axonal guidance during the development of the nervous system is thought to be highly Hernaiz-Llorens M, Pascual M, regulated through interactions of transmembrane receptors with attractive, repulsive, and Muhaisen A, Navarro X, del Valle J trophic cues. Similar mechanisms regulate axonal regeneration after injury. The transected axon and Soriano E (2019) Cholesterol undergoes morphological changes to form the growth cone, a highly dynamic structure that Depletion Regulates Axonal Growth and Enhances Central and Peripheral senses the environment and leads the regenerative growth (Allodi et al., 2012). Membrane Nerve Regeneration. receptors localized in the growth cone have an important role in axonal signaling (Guirland Front. Cell. Neurosci. 13:40. et al., 2004; Kamiguchi, 2006). The regenerative shift of axotomized neurons is promoted doi: 10.3389/fncel.2019.00040 by injury-induced signals, which stimulate the transcription of various trophic factors, Frontiers in Cellular Neuroscience | www.frontiersin.org 1 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration adhesion molecules, growth-associated proteins and structural Complex (RPN1051, GE Healthcare); and Streptavidin-Alexa components needed for axonal regrowth and cell survival (Rishal Fluor 594 (S32356, Thermo Fisher). and Fainzilber, 2014). The following drugs and reagents were used: poly-D-Lysine Many intrinsic and extrinsic signals can promote or inhibit (P7280, Sigma); laminin (L2020, Sigma); Nystatin dihydrate axonal regeneration. Due to the importance of these signals (N4014, Sigma); Cholesterol Oxidase Streptomyces sp. (ChOx) during axonal degeneration and regeneration after peripheral (228250, Calbiochem); Methyl-β-cyclodextrin (MβCD) (C4555, nerve injury, microdomains in the membrane that cluster a range Sigma); DMSO (D5879, Sigma); phalloidin – TRITC (P1951, of proteins and molecules related to cellular signaling may play a Sigma); biocytin (B4261, Sigma); Cholera Toxin Subunit key role in the regulation of these pathways. In particular, lipid B (Recombinant) Alexa Fluor 594 (CTxB-594) (C34777, rafts have been described as cholesterol-enriched cell membrane Life BioSciences). microdomains that compartmentalize lipids and protein to form signaling platforms (Golub et al., 2004). Primary Cultures Cholesterol is a major component of the nervous system, Hippocampus being essential for normal brain development. The brain is the Primary cultures of mouse hippocampi were prepared from E16- most cholesterol-rich organ, containing about 20% of the body’s E17. Pregnant CD1mice were sacrificed by cervical dislocation, total cholesterol (Bjorkhem and Meaney, 2004). Under normal and the fetuses were collected in a PBS-glucose 0.3% solution physiological situations and because plasma lipoproteins do not and then decapitated. Hippocampi were isolated and trypsinized cross the intact blood-brain barrier, nearly all cholesterol in the for 6 min at 37◦ C. Trypsin was then neutralized with FBS brain is synthesized in situ (Dietschy and Turley, 2001). Brain and incubated with DNase for 10 min at 37◦ C. Neurons cholesterol is an important structural component of cellular were then centrifuged at 800 rpm for 5 min, resuspended membranes and myelin. It is also required for the synthesis of and plated in pre-coated culture glasses with poly-D-lysine in steroid hormones and for the organization of lipid rafts, which medium containing Neurobasal (w/o L-glutamine, w/Phenol are involved in many aspects of brain function, such as growth Red; GIBCO, 21103-049), 1% penicillin/streptomycin (GIBCO, factor signaling, synapse and dendritic formation (Goritz et al., 15140-122), 1% glutamine (GIBCO, 25030-024) and 2% B27 2005), and axon elongation and guidance (de Chaves et al., 1997). (GIBCO, 17504-044). Here we studied the effects of altered membrane integrity by reducing the cholesterol content in the axons of three neuronal Cerebellum systems, namely hippocampal and cerebellar external granular Primary cultures of cerebellums were prepared from P4–P5 layer (EGL) cells as a Central Nervous System (CNS) example, CD1 mice sacrificed by decapitation. Cerebellums were isolated, and the dorsal root ganglion (DRG) neurons as a Peripheral mechanically disaggregated and trypsinized as previously Nervous System (PNS) example. We show that depletion of described. After centrifugation, neurons were resuspended in cholesterol leads to increased sizes of growth cones, filopodial 2 mL of DMEM, and EGL were isolated by centrifugation extensions and neurite length. Moreover, we also demonstrate in a percol gradient. After a wash centrifugation, EGL that cholesterol membrane and raft disruption increase the were plated in pre-coated culture glasses with poly-D-lysine regenerative capacity of axons after axotomy both in vitro and in medium containing DMEM (GIBCO, 41966-029), 1% in vivo and enhance muscle and sensory re-innervation of penicillin/streptomycin (GIBCO, 15140-122), 1% glutamine distal targets. On the basis of our findings, we propose that (GIBCO, 25030-024), 4.5% D-(+)-Glucose (Sigma, G-8769), 5% acute reduction of neuronal cholesterol emerges as a potential NHS (GIBCO, 26050-088), and 10% FBS (GIBCO, 16000-044) therapeutic strategy to improve regenerative outcomes after for 24 h, and then NHS and FBS were replaced by 2% B27 peripheral nerve lesion. (GIBCO, 17504-044) and 1% N2 (GIBCO, 17502-048). Dorsal Root Ganglion (DRG) MATERIALS AND METHODS Primary cultures of DRG neurons were prepared from E13–E14 mice. Pregnant CD1 mice were sacrificed by cervical dislocation, Reagents and Antibodies and the fetuses were collected and decapitated. DRG neurons The following antibodies were used: anti-GFP (A11122, were isolated and trypsinized as previously described. After Invitrogen); anti-IIIβ-tubulin (MMS-435P, Covance); anti- centrifugation, neurons were resuspended and plated in growth associated protein 43 (GAP43) (AB5220, Millipore); pre-coated culture glasses with poly-D-lysine and laminin anti-myelin basic protein antibody (MBP) (ab7349, Abcam); in medium containing DMEM (GIBCO, 41966-029), 1% anti-neurofilament H (NF-H) (AB5539, Millipore); Donkey anti- penicillin/streptomycin (GIBCO, 15140-122), 1% glutamine Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (GIBCO, 25030-024), 0.06% D-(+)-Glucose (Sigma, G-8769), Alexa Fluor 488 (A-21202, Thermo Fisher); Donkey anti-Rabbit 0.0045% NaHCO3 (GIBCO, 25080-060), 2% B27 (GIBCO, IgG (H+L) Highly Cross-Adsorbed Secondary Antibody Alexa 17504-044) and 5 µg/ml NGF. Fluor 488 (A-21206, Thermo Fisher); Goat anti-Chicken IgY Organotypic entorhino-hippocampal slice cultures were (H+L) Alexa Fluor 488 (A-11039, Thermo Fisher), Biotinylated obtained from P0 actin-GFP and WT mice sacrificed by Horse anti-rabbit IgG (BA-1000, Vector); Biotinylated Goat decapitation. Horizontal sections 325 µm thick containing anti-rat IgG (BA-9400, Vector), Streptavidin-Biotinylated HRP both the entorhinal cortex (EC) and the hippocampus were Frontiers in Cellular Neuroscience | www.frontiersin.org 2 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration obtained by cutting tissue pieces in a chopper (McIlwain Tissue exposed at the midthigh and carefully freed from adherences Chopper, Standard Table, 220V, Prod 10180-220). Slices were to surrounding tissues. Afterwards, it was cut at 45 mm from laid on a porous Millicell CM plate culture insert (Millipore, the tip of the 3rd toe and immediately repaired using two PICM03050) and incubated using the interface culture technique. epineurial sutures (10–0), maintaining the fascicular alignment The medium comprised 35.5% Neurobasal, 25% MEM powder, of the sciatic branches. Finally, the wound was sutured in planes 25% NHS (GIBCO, 26050-088), 12.5%HBSS (w/Mg, w/Phenol and disinfected. Animals were left to recover on a hot pad after Red, GIBCO, 24020-083), 0.5% D-(+)-glucose (Sigma, G-8769), being returned to their cages. The left paw was left uninjured 1% glutamine (GIBCO, 25030-024), 1% penicillin/streptomycin as a control. (GIBCO, 15140-122), 1.1% sodium pyruvate (Sigma, P2256- All the animals had food and water ad libitum and were 5G), 0.04% NaHCO3 (GIBCO, 25080-060), 2% B27 (GIBCO, kept at a standard temperature (22 ± 2◦ C) and under 12:12-h 17504-044) and 1% N2 (GIBCO, 17502-048). The medium was light-dark cycles (300 lux/0 lux). The experimental procedures changed every 2 days. After 21 days in vitro (DIV), axotomy followed the recommendations of the European Communities was performed between the EC and the hippocampus with a Council Directive 2010/63/EU for the care and use of laboratory needle, and WT hippocampi were put with EC GFP. The cultures animals and were approved by the Committee for Ethics on were treated for 10 days with Nystatin or control medium Experimental Animal and Human Research of the Universitat and fixed with 4% paraformaldehyde (PFA) for 1h. Cultures Autònoma de Barcelona. were cut into 60 µm slices with a vibratome (Leica VT 1000 S) and cryopreserved at −20◦ C until immunohistochemistry Electrophysiology Tests was performed. To test possible effects of MβCD on intact animals, electrophysiological tests were performed every 7 days after Drug Treatments in vitro beginning of the treatment and for 4 weeks. To monitor Growth Cone and Filopodium Experiments peripheral nerve regeneration, electrophysiological tests were Hippocampal and EGL primary cultures were treated after 3 DIV performed at 14, 21, 25, 28, and 33 days post-injury (dpi). and DRG after 1 DIV with 2.5 µg/mL Nystatin for 10 min, Animals were anesthetized with pentobarbital (10 mg/kg) and 0.5 mM MβCD for 10 min or 2U ChOx for 2 h. They were then the sciatic nerve was stimulated using two needle electrodes incubated for an additional 30 min in culture media (Neurobasal inserted percutaneously at the sciatic notch, applying single or DMEM) and then fixed in 4% PFA. rectangular pulses of 0.02 ms up to the voltage required to obtain a maximal evoked response. The compound muscle action Axon Extension and Branching Experiments in DRG potentials (CMAP, M wave) evoked by stimulation of motor Neurons nerve fibers were recorded from the tibialis anterior (TA) and After 2 h in culture, 2.5 µg/mL Nystatin and 2U ChOx were plantar (PL) muscles with microneedle electrodes. All potentials added to the media for 24 h and neurons were then fixed. After were amplified and displayed on a digital oscilloscope (Tektronix 22 h in vitro, and therefore 2 h before neurons were fixed, 0.5 mM 450S; Tektronix, Beaverton, OR, United States) at the appropriate MβCD was added to the media. settings for latency and amplitude measurements from the baseline to the maximal negative peak. During the tests, the Axotomy Experiments animals were placed over a warmed flat coil controlled by a hot Axotomy in microfluidic chambers (AX50010TC, Millipore) water circulating pump to maintain body skin temperature. was performed with a pipette connected to a vacuum pump. Complete axotomy was verified in the inverted microscope. Locomotion Evaluation Immediately after axotomy, 2.5 µg/mL Nystatin was added and The DigiGait system (Mouse Specifics, Boston, MA, maintained in the culture for 3 days. In organotypic cultures, United States) was used to assess locomotor performance 5 µg/mL Nystatin was added to the media just after axotomy and of healthy animals treated with MβCD or vehicle at weekly maintained for 10 days, changing the media every 2 days. intervals during treatment. The mice were forced to run over the transparent belt of a motorized treadmill and recorded with Animals and Surgical Procedures a high-speed video camera (80 frames/s) from below while To study the effect of cholesterol depletion on healthy mice, running at a constant treadmill velocity of 20 cm/s. A minimum 10 OF1 female mice (20–25 g) were divided in two groups of 200 images were collected for each walking mouse containing and treated with saline (vehicle) or MβCD 1000 mg/kg/week more than eight strides for each run and each video was analyzed intraperitoneal (i.p.) during 1 month. To study the effect of with the DigiGait software. The print length, and the toe spread lipid raft disruption on peripheral nerve regeneration, 13 female distance between toes 1–5 and toes 2–4 were measured, and the mice (25–30 g) were injured on the sciatic nerve and allowed sciatic functional index (SFI) between the right and the left paw to regenerate for 1 month while receiving saline (n = 6) or was calculated (Navarro, 2016). 1000 mg/kg/week MβCD (n = 7) i.p. treatment. To perform the nerve injury, animals were anesthetized by Mechanical Algesimetry i.p. injection of ketamine (90 mg/kg; Imalgene 500, Rhône- The algesimetry tests for mechanical stimuli were performed Merieux, Lyon, France) and xylazine (10 mg/kg; Rompun, Bayer, on both hindpaws every week along treatment in uninjured Leverkusen, Germany). The right sciatic nerve was surgically animals. Sensibility to a non-noxious mechanical stimulus was Frontiers in Cellular Neuroscience | www.frontiersin.org 3 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration measured by using an electronic Von Frey algesimeter (Bioseb, For cholesterol depletion analysis, samples were defrosted for Chaville, France). Mice were placed on a wire net platform in 30 min at room temperature and then rehydrated with PBS plastic chambers 10 min before the experiment for habituation. for 5 min. DRG neurons were then incubated for 3 h at room The hindpaw plantar surface was stimulated from the bottom temperature with CTxB (1:200) in PBS. Slides were washed and of the box by applying a 0.4 mm non-noxious pointed metal mounted using Mowiol. probe to the central area of the paw, and then slowly increasing For immunohistochemistry, samples were defrosted at room the pressure until the mouse raised the paw, with a 35 g cut temperature, blocked with PBS-specific animal serum, and off force to avoid skin damage. The mechanical nociceptive incubated overnight at 4◦ C with the primary antibodies rabbit threshold was taken as the mean of three measurements per anti- GAP43 (1:100), rat anti- MBP (1:200) and chicken anti- paw and the threshold was expressed as the force (in grams) NF-H (1:100). After washes, sections were incubated with the at which mice withdrew the paw in response to the stimulus respective biotinylated secondary antibody (1:200) for GAP43 (Cobianchi et al., 2014). and MBP and then for 1 h at room temperature with conjugated Alexa Fluor streptavidin (1:200) or Alexa Fluor anti-chicken IgY. Pinprick Test Finally, sections were mounted with Mowiol containing DAPI for Progression of nociceptive reinnervation of the hindpaw was nuclear staining. assessed at 14, 21, 25, 28, and 33 dpi by means of the pinprick test. Awake animals were gently wrapped in a cloth with the injured Image Analysis paw facing upward. The skin surface near C plantar pad and 4th Images of primary cell cultures were taken in an epifluorescence toe (more distal) were stimulated with a blunt needle with enough microscope (Eclipse Nikon E1000) with a 60× oil-immersion intensity to indent the skin without damaging it (Cobianchi objective. To take images from the in vitro axotomy experiments, et al., 2014). Each site was stimulated twice and responses were we used an inverted microscope (Olympus ScanR) with a recorded as positive only when clear pain reactions such as fast 20× objective. For images of the organotypic cultures, we withdrawal or vocalization were triggered. Positive responses used a confocal microscope (Leica TCS SP5) with a 20× were taken as a sign of functional reinnervation of the skin. objective and a 20× objective with 2× zoom to quantify axon regeneration. Images of the tibial nerve with MBP and NF-H immunostaining were taken10 mm distally from the injury site Histology, Immunohistochemistry and with an epifluorescence microscope (Eclipse Ni, Nikon) attached Immunocytochemistry to a digital camera (DS-Ri2, Nikon). Finally, we used a confocal Neurons were fixed with a solution of 4% PFA in PBS for 10 min. fluorescent microscope (Zeiss LSM 700) to acquire the images Subsequently, they were rinsed with PBS and permeabilized with from the SC and DRG tissue sections stained with GAP43, and a solution of triton 0.1% X-100 in PBS for 10 min. Afterwards, images were analyzed with ImageJ (Schneider et al., 2012). Briefly, they were rinsed with PBS once more, and blocking solution the area of the soma of at least 100 DRG neurons/animal and (NHS 10% in PBS) was added for 1 h at room temperature. After 10 motoneurons/animal were taken as ROI, and the integrated blocking, the cells were incubated for 2 h with the respective density (mean gray value × area) was obtained. The mean value primary antibodies diluted in blocking solution. Unbound obtained for each animal was used for comparison (Romeo- primary antibodies were washed with PBS, and neurons were Guitart et al., 2018). incubated with the respective secondary antibodies in blocking solution for 1 h at room temperature. For F-actin staining Statistical Analysis after permeabilization, phalloidin diluted in PBS was added and All in vitro experiments were done three times independently. maintained for 30 min. Neurons were mounted using Mowiol. Data show means ± SEM. To analyze the statistics, we used Organotypic slices were rinsed with PBS with 0.5% Triton GraphPad software and applied ANOVA test for multiple three times, and were incubated for 2 h at room temperature with conditions or the Student’s t-test for two conditions. In vivo blocking solution (10% NHS, 0.2M Glycine in PBS-Gelatin with results are presented as mean ± SEM and means were compared 0.5%Triton). After blocking, the cells were incubated overnight with two-tailed, unpaired Student’s t-test, two-way ANOVA at 4◦ C with the respective primary antibodies diluted in antibody followed by Tukey’s Multiple Comparison Test or Mantel–Cox solution (5% NHS in PBS-Gelatin with 0.5%Triton). Unbound test. Differences were considered significant at p < 0.05. primary antibodies were washed with PBS with 0.5% Triton, and slices were incubated with the respective secondary antibodies in antibody solution for 2 h at room temperature. RESULTS For the in vivo regeneration experiment, mice were transcardially perfused with 4% PFA in PBS (0.1 M, pH 7.4) 1 month after beginning the treatment. Subsequently, the Cholesterol Depletion Increases Growth sciatic nerve, spinal cord, L4 and L5 DRG neurons were removed, Cone Area and Number of Filopodia in postfixed for 24 h with PFA and cryopreserved in PB containing Hippocampal and EGL Cerebellar Axons 30% of sucrose for immunohistochemistry analysis. Samples Growth cones play a key role during axonal growth and were serially cut (15 µm) with a cryotome (Leica CM190) and guidance. First, we examined the effect of cholesterol depletion stored at −20◦ C for further analysis. on the morphological features of growth cones. To this end, Frontiers in Cellular Neuroscience | www.frontiersin.org 4 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration we treated hippocampal and EGL neurons with two drugs that depletion induced by these drugs led to a significant increase efficiently reduce cholesterol in the cell membrane (Nystatin in the number of filopodium-like structures in both types of and MβCD), and with ChOx, a cholesterol-depleting enzyme neuron (Figures 2C,D). (Figure 1) (Bolard, 1986; Cahuzac et al., 2006; Zidovetzki and These results indicate that a reduction in the cholesterol Levitan, 2007). Both neuron types were kept in culture for content of the cell membrane of both types of neuron promoted 3 DIV before being treated with the aforementioned drugs. In an increase in the number of filopodium-like structures hippocampal neurons, depletion of cholesterol driven by the and branching. In contrast to the effect of these drugs on three drugs significantly increased the growth cone area identified growth cone area, these compounds enhanced the number of by phalloidin staining (Figures 1A,C). However, only Nystatin filopodia/branches in hippocampal and EGL neurons. increased this parameter in EGL neurons (Figures 1B,D). Overall, our results show that cholesterol depletion increases the growth cone area in hippocampal and EGL neurons in the CNS. Cholesterol Depletion Affects Growth During the course of the experiments, we observed another Cone Area, Number of Filopodia, and effect of cholesterol depletion, namely the development of Neurite Extension in DRG Neurons numerous filpodium-like extensions/branching points along To determine whether the effects of cholesterol depletion neurites. Filopodia, dynamic membranous structures driven observed in the CNS neurons can be extrapolated to the PNS, we from the actin cytoskeleton, are necessary for axonal motility, performed the same experiments using DRG neurons (Figure 3). guidance, branching and regeneration. To determine the DRG neurons were isolated from mouse embryos at E13–14 and importance of cholesterol in the formation of filopodium- treated after 1 DIV with cholesterol removal agents. Treatment like structures, we again treated hippocampal and EGL with Nystatin and ChOx led to an increase in growth cone neurons with Nystatin, ChOx, and MβCD (Figure 2). The area in DRG neurons (Figures 3A,C), and the removal of filopodium-like structures were counted in discrete segments cholesterol with any of the three drugs enhanced the number of of hippocampal and EGL axons (Figures 2A,B). Cholesterol filopodia (Figures 3B,D). FIGURE 1 | Cholesterol depletion increases growth cone area in hippocampal neurons and EGL neurons. Examples of hippocampal (A) and EGL (B) growth cones treated with control culture media, 2.5 µg/ml Nystatin, 2U ChOx, and 0.5 mM MβCD and stained with phalloidin. Growth cones areas from three independent experiments were measured. Data represent means ± SEM (C,D). N = 150–250 growth cones in each condition (One-way ANOVA, Dunnett’s Multiple comparison test ∗∗∗ p < 0.0001). Scale bar 5 µm. Frontiers in Cellular Neuroscience | www.frontiersin.org 5 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration FIGURE 2 | Cholesterol depletion increases filopodium-like structures in hippocampal neurons and EGL neurons. Examples of hippocampal (A) and EGL (B) filopodium-like structures in axons treated with control culture media, 2.5 µg/ml Nystatin, 2U ChOx, and 0.5 mM MβCD and stained with phalloidin. The density of filopodium-like structures was measured in discrete segments of axons in three independent experiments. Data represent means ± SEM (C,D). N = 150–250 neurons in each condition (One-way ANOVA, Dunnett’s Multiple comparison test ∗∗ p < 0.01, ∗∗∗ p < 0.0001). Scale bar 5 µm. We also quantified the effect of cholesterol depletion on attributed mainly to the generation of an axon blocking “milieu.” neurite extension and branching (Figure 4). Treatments were As we have shown that cholesterol depletion increases axonal performed for 24 h in the case of Nystatin and ChOx and growth cone area and axonal extension, we next addressed, using for 2 h before fixation in the case of MβCD, as we observed an in vitro and an ex vivo system, whether depletion of this lipid increased cell death with longer incubations with this drug. Only enhances axonal regeneration. the treatment with Nystatin increased the total length of DRG First, E16 hippocampal neurons were seeded in a microfluidic neurites while ChOx and MβCD did not affect this parameter chamber (Figure 5) and neurons were axotomized. This chamber (Figures 4A,B). We found that 24 h treatments with Nystatin and allowed us to differentially treat the axons, on one side with ChOx led to a significant increase in the number of branching control medium and on the other with medium supplemented points (Figures 4A,C). with Nystatin applied immediately after axotomy (Figure 5D). These results suggest that pharmacological removal of Axons treated with Nystatin showed a significant increase in cholesterol from the cell membrane has similar effects on CNS length when compared with those cultured in basal control (hippocampal and cerebellar) and PNS (DRG) neurons. An acute medium (Figures 5A–C). decrease in cholesterol levels per se increases growth cone area These results suggest that the removal of cholesterol by and filopodia/branching point density in developing neurons. means of Nystatin enhances axon regeneration after axotomy in vitro. We next used the ex vivo organotypic model of axotomy and regeneration (del Rio and Soriano, 2010). Slice Cholesterol Depletion Improves Axonal co-cultures from P0 mouse hippocampi and the EC were Regeneration After Axotomy in vitro and isolated and kept in culture for 21 days (Figure 6). Some ex vivo EC slices were obtained from actin-GFP transgenic mice. At One of the hallmarks of CNS regeneration is the difficulty 21 DIV, axotomy was performed sectioning the entorhino- to achieve the regrowth of damaged axons. This difficulty is hippocampal connection (EHP) using a needle, and mixed Frontiers in Cellular Neuroscience | www.frontiersin.org 6 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration FIGURE 3 | Cholesterol depletion affects growth cone area and the density of filopodia in DRG neurons. Examples of DRG growth cones (A) and filopodia (B) incubated with control media, 2.5 µg/ml Nystatin, 2U ChOx, and 0.5 mM MβCD and stained with phalloidin. Growth cone areas and filopodia from three independent experiments were measured. Data represent means ± SEM (C,D). N = 150–250 neurons in each condition (One-way ANOVA, Dunnett’s Multiple comparison test ∗∗ p < 0.01, ∗∗∗ p < 0.0001). Scale bar 5 µm. co-cultures were generated. The hippocampi from actin-GFP Treatment With MβCD Has No Effect on co-cultures were removed and replaced by hippocampi from Healthy Mice WT mice organotypic cultures, thereby allowing the direct The results of motor electrophysiological tests performed visualization of regenerating GFP-positive EC-hippocampal showed that MβCD administration in vivo did not cause any axons. Axotomized organotypic cocultures were treated with impairment in the mice. The M wave amplitude (Supplementary Nystatin for 10 additional days (Figure 6A). In axotomized Figures S1A,B) and latency (Supplementary Figures S1C,D) co-cultures that were not treated with Nystatin, virtually of TA and PL muscles did not present significant differences no regenerating axons (or very few) were found in the between MβCD treated and saline control groups. The walking SLM/ML layers, which are the layers of termination of the track test yielded no differences in the SFI between groups EHP pathway (Figures 6B,D). In contrast, after Nystatin (Supplementary Figure S1E). Finally, mechanical algesimetry treatment, we observed a 10-fold increase in the density was performed to evaluate possible changes in nociception in of regenerating EC axons present in the SLM/ML layers MβCD treated animals. Results showed no significant differences (Figures 6C,D). Organotypic cultures treated with Nystatin between control and treated animals in pain threshold along time showed a normal morphology and distribution, and no evidence (Supplementary Figure S1F). of neuronal degeneration was appreciated (Figure 6C). In another set of experiments, WT EC slice-cocultures were generated and axotomized as described above (Figure 6A) and Treatment With MβCD Alters the Integrity regenerating EC axons were visualized by Biocytin injections of the Lipid Raft by Reducing the (del Rio and Soriano, 2010). Again, the number of regenerating Amount of Membrane Cholesterol EHP axons was markedly increased by the incubation with Our previous results suggest that cholesterol depletion is directly Nystatin. Together with the above results, the present findings involved in promoting axonal regeneration. However, we next indicate that cholesterol depletion favors axonal regeneration sought to determine the effect of acute reduction of cholesterol in vitro and ex vivo. levels in the in vivo model of peripheral nerve lesion and Frontiers in Cellular Neuroscience | www.frontiersin.org 7 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration FIGURE 4 | Cholesterol depletion increases neurite elongation and branching in DRG neurons. Examples of DRG neurons incubated for long periods with control media, 2.5 µg/ml Nystatin, 2U ChOx, and 0.5 mM MβCD (A) and immunolabeled with anti-βIII-tubulin. Total neurite extension and number of branching points in each neuron were quantified in each condition in three independent experiments. Data represent means ± SEM (B,C). N = 30–60 neurons in each condition (One-way ANOVA, Dunnett’s Multiple comparison test ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.0001). Arrows point cell body. Scale bar 100 µm. regeneration. Cholesterol is a major component of lipid rafts, the neuronal regenerative program. No significant difference which are specialized membrane microdomains that hold a was observed for motoneurons (Figure 8D). Moreover, myelin multitude of signaling receptors. It has recently been shown that staining of the regenerated nerve was conducted to assess disruption of the raft by acute depletion of cholesterol promotes whether lipid raft disruption could affect axonal remyelination. regeneration and functional recovery after spinal cord injury Qualitative assessment of both saline- and MβCD-treated (Tassew et al., 2014). Thus, we first examined the integrity of animals (Figure 8E) shows similar presence of myelin in nerve raft structures in DRG nerves after acute depletion of cholesterol fibers of both groups, thus indicating that lipid raft disruption did by treatment with MβCD. Contralateral DRG neurons of injured not block remyelination after injury. mice treated with MβCD or saline were incubated with the CTxB. This fraction of the toxin has a known affinity for ganglioside GM1, a lipid raft-associated molecule (Schnaar et al., 2014) widely Cholesterol Depletion Induced by MβCD used as a marker of the presence of cholesterol in the cellular Improves Muscle Reinnervation and membrane and of lipid raft integrity. Fluorescent staining showed Sensory Responses in Mice clear CTxB labeling in the plasma membrane of DRG neurons Electrophysiological tests performed to follow up nerve from animals that received vehicle for 1 month (Figure 7A) in regeneration after sciatic nerve section and repair showed a comparison with those treated with MβCD (Figure 7B). progressive increase in the amplitude of the M wave of the two muscles in both groups, thereby indicating progressive reinnervation of the muscles by the regenerated axons. Although Cholesterol Depletion Induced by MβCD no differences were seen for the proximal TA muscle, MβCD- Increases GAP43 in Sensory Neurons treated mice had significantly higher M wave amplitude of the After Axotomy PL muscle than control animals at 33 days (Figures 9A,B), To study the regenerative response in sensory (Figure 8A) and indicative of greater reinnervation of the distal muscles. Values motor (Figure 8B) neurons after nerve section, the expression of M wave latency did not show significant differences between of the GAP43 was measured. GAP43 is expressed at high levels the two groups (Figures 9C,D). during development and axonal regeneration, and it is considered No signs of autotomy, that could be indicative of neuropathic a crucial component of an effective regenerative response in the pain, were found in any studied mice. Recovery of sensitivity nervous system, being used as a marker of regeneration in injured was studied by means of the pinprick test on areas of the paw axons. The results showed higher immunoreactivity for GAP43 in from proximal to distal region of the sciatic nerve innervation DRG neurons in injured animals treated with MβCD compared territory (Cobianchi et al., 2014). In the C plantar pad, no to those treated with vehicle (Figure 8C). This observation animals showed positive response at 21 days, and with time indicates that cholesterol depletion after sciatic injury enhances more animals recovered sensory response without differences Frontiers in Cellular Neuroscience | www.frontiersin.org 8 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration FIGURE 5 | Nystatin improves axon regeneration after axotomy in vitro. Examples of hippocampal axon regrowth at 24, 48, and 72 h after axotomy in control condition (A) and in response to treatment with 2.5 µg/ml Nystatin (B). Axon lengths were measured in each condition. Data represent means ± SEM (C). Schematic diagram of a microfluidic chamber (D). Cell bodies are in the central channel (white), axon growth through microfluidic channels (blue) to both sides (green and red). After axotomy, control medium was applied on one side (red) and Nystatin on the other (green). N = 3 chambers (t-Test in each time point, ∗∗∗ p < 0.0001). Scale bar 50 µm. Frontiers in Cellular Neuroscience | www.frontiersin.org 9 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration FIGURE 6 | Nystatin increases EC axon regeneration in organotypic cultures. (A) Schematic diagram that summarizes the experimental protocol. Slice co-cultures from P0 actin-GFP and WT mice were isolated, as described in Section “Materials and Methods” and kept in culture 21 DIV. Axotomy of the EHP was performed, and slices were treated for 10 DIV with control medium or 5 µg/ml Nystatin. (B) Representative examples of control hippocampal slices show virtually no regenerative axons (upper panel) or very few regenerative axons (lower panel). (C) Examples of hippocampal slices treated with Nystatin show moderate axonal regeneration (upper panel) or robust axonal regeneration (lower panel). Axons that cross a 250 µm line were counted in three random fields near the dentate gyrus in each slice. Data represent means ± SEM (D). One dot corresponds to one slice (t-Test ∗ p < 0.05). Scale bar 100 µm. ML, molecular layer; GL, granule cell layer; H, hilus. between groups. However, in the 4th toe, which is the most ensure survival and regeneration. Moreover, a growth cone distal area, cholesterol-depleted mice showed positive response is formed at the tip of growing axons, and this process earlier than injured controls, and at 33 dpi, 50% of the MβCD requires morphological and biochemical changes in the plasma group showed positive responses whilst only 12.5% of the vehicle- membrane, including membrane extension and remodeling. In treated animals did (Figures 9E,F). this context, cholesterol-rich lipid rafts, also called membrane microdomains, play a key role as signaling platforms of both growth-promoting and growth-inhibiting molecules. With DISCUSSION regard to the possible contribution of lipid rafts and cholesterol to nerve regeneration, conflicting views can be found in the Following axotomy, several metabolic changes occur in literature. Some authors demonstrated that lipid rafts promote injured neurons to reinnervate denervated targets and thus axonal regeneration (Santuccione et al., 2005; Zhang et al., 2013) Frontiers in Cellular Neuroscience | www.frontiersin.org 10 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration FIGURE 7 | MβCD disruption of lipid rafts in DRG neurons. (A,B) Representative images of the DRGs of animals treated with vehicle (A) and MβCD (B). Note the absence of CTxB membrane staining in B after MβCD treatment, indicating lipid raft disruption. Scale bar: 100 µm. while others claim that they have an inhibitory role (Vinson Next, we addressed whether cholesterol depletion increases et al., 2003; Hausott et al., 2011). These contradictions can be the regrowth of axotomized neurons in two in vitro models: explained by the fact that, in addition to a variety of proteins and dissociated hippocampal cultures and axotomized EC- molecules that promote axonal regeneration, such as receptors hippocampal slice cultures. In both cases, we found a marked of neurotrophic factors, lipid rafts also contain proteins and increase in the regenerative potential of lesioned axons, molecules that exert an inhibitory role, such as receptors of again suggesting that cholesterol depletion not only increases myelin-associated proteins (Kappagantula et al., 2014). On the axonal length in developing axons but also axonal regrowth other hand, there is also some discrepancy about the role of and regeneration in vitro. In addition, the observation that lipid rafts in cell apoptosis. It is described that pro-apoptotic regenerated EC-hippocampal axons were correctly targeted to receptors like Neogenin, FAS and its ligand, JNK, PKC, Src the appropriate termination layers (SLM and ML) indicates kinases and a wide variety of lymphocyte-related receptors are that cholesterol depletion does not alter the molecular more functional when are localized in lipid rafts, inducing a line mechanisms involved in the guidance and targeting of these of investigation in which lipid rafts are targets for chemotherapy axons to the hippocampus. (George and Wu, 2012). However, receptors related with cell Membrane extension during axon growth after axotomy survival, like Akt cascade, also depend on lipid rafts (George requires the incorporation of new lipids to the tips of the and Wu, 2012). It has been shown that cholesterol depletion regenerating axons. Incorporated cholesterol in the axons of with MβCD protects cerebellar neurons from apoptosis (Zhou peripheral neurons after axotomy comes from transported et al., 2012) and after spinal cord injury, lipid raft disruption cholesterol synthesized in the cell bodies of lesioned neurons promotes cell survival due to a loss of function of Neogenin (de Chaves et al., 1997), and from recycled cholesterol from (Tassew et al., 2014). In conclusion, some of these studies cellular and myelin debris originated during the injury were performed in vitro and sought to address the cellular (Goodrum, 1991; de Chaves et al., 1997). The existence of pathway or the signaling of a specific neurotrophin through multiple complementary mechanism to incorporate cholesterol lipid rafts, and did not take into account all the trophic factors into the re-growing membranes suggest a redundancy in the and cell responses that occur in vivo, but instead focused on process of cholesterol reutilization. Our results in vitro and single pathways. in vivo suggest that acute and medium-term reduction of In the present study, we show that cholesterol depletion in cholesterol levels have positive effects for axon elongation and juvenile cultured neurons increases the size of growth cones and regeneration after nerve injury. However, further studies are enhances neurite and axonal extension and the marked formation required to evaluate long-term effects of systemic cholesterol of filopodium-like extensions in both central and peripheral reduction. Secondary undesired effects associated with alteration neurons. Although we did not address the underlying molecular of membrane cholesterol levels should also have to be considered. mechanisms, we believe that both the membrane fluidity induced Previous reports have found that MβCD may alter crayfish by cholesterol depletion and partial lipid raft disruption may neuromuscular junction functioning in cold conditions (14◦ C) account for the effects observed, as extracellular matrix and but not at 21◦ C (Ormerod et al., 2012) and cholesterol depletion adhesion receptors are also grouped in lipid rafts (Leitinger in Caenorhabditis elegans results in decreased motility (Merris and Hogg, 2002; Decker and ffrench-Constant, 2004; Head et al., 2004). Moreover, reduction of membrane cholesterol et al., 2014). In addition, the increased size of neurites suggests alters ion channel activity in sensory peripheral neurons (Saghy that cholesterol depletion might be involved in the positive et al., 2015), with direct consequences for their hypersensitivity regulation of the exocytotic machinery needed for neurite growth (Pristera et al., 2012) and the development of neuropathic (Ros et al., 2015). pain (Ferrari and Levine, 2015; Amsalem et al., 2018). As a Frontiers in Cellular Neuroscience | www.frontiersin.org 11 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration FIGURE 8 | MβCD increases GAP43 expression in injured DRG neurons. (A,B) Detail of DRG (A, dashed line) and motoneurons (B, dashed line) analyzed for GAP43 expression. (C,D) Quantification of GAP43 positivity in DRG neurons (C) and motoneurons (D) from animals treated with vehicle or MβCD vs. Saline. (E) Detail of a regenerated nerve stained for cell nuclei (blue), NF-H (green), and GAP43 (red) of saline- (top) and MβCD-treated (bottom) animals. Scale bar: 15 µm in (A,B); 50 µm in (E). Student’s t-test, ∗∗ p < 0.01. consequence, electrophysiological tests, pain threshold tests 2016). Of particular interest, the algesimetry results showed no and locomotion studies were performed on healthy animals to indication of hyperalgesia in intact mice, and the absence of evaluate any possible effect derived from the systemic MβCD autotomy in mice in the regeneration experiment also points administration and the consequent membrane cholesterol out that there was no increased pain sensitivity after lipid raft depletion. Our results on healthy mice showed no significant disruption (Casals-Diaz et al., 2009). Therefore, although some differences between treated and control animals with reported uncontrolled effect cannot be dismissed, 1 month of systemic values similar to those found in previous studies in the MβCD administration and lipid raft disruption did not alter literature (Verdu et al., 1996; Bruna et al., 2010; Ale et al., normal nervous system function in mice. Frontiers in Cellular Neuroscience | www.frontiersin.org 12 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration FIGURE 9 | MβCD treatment accelerates motor and sensory functional recovery following sciatic nerve injury. (A–D) Electrophysiological results after nerve injury in mice treated with vehicle or with MβCD. Results of CMAP amplitude (A,B) and latency (C,D) recorded in TA and PL at 14, 21, 25, 28, and 33 dpi. Data are shown as the mean and SEM (bars). ∗ p < 0.05 with two-way Anova and Bonferroni test. (E,F) Pinprick test results to analyze sensory reinnervation in the hindpaw pad C (E) and 4th toe (F). Results show the percentage of animals with positive response in each area in the groups treated with vehicle or MβCD. p < 0.05 Mantel–Cox test indicate differences in the proportion of mice with reinnervation. We then studied the effect of cholesterol depletion on amphipathic properties, and molecules can be encapsulated peripheral nerve regeneration. To this end, we chose an in vivo inside the cavity of cyclodextrins. Thus, MβCD may act not model of peripheral nerve injury to avoid focusing on a single only by extracting cholesterol from the plasma membrane and pathway or molecule, thus gaining a complete view of nerve disrupting lipid rafts but also by catalyzing neurotrophic factors regeneration. Cholesterol depletion and lipid raft disruption and favoring their uptake by neurons or glial cells that play a were achieved by means of treatment with MβCD (Zidovetzki role in peripheral nerve regeneration. This could explain the and Levitan, 2007), a cyclic oligosaccharide that has been increase in the number of GAP43-positive DRG neurons, an used as an antineoplastic (Grosse et al., 1998) and also as a observation that indicates that, after MβCD treatment, there complexing agent in pharmacological applications (Upadhyay are more neurons able to regenerate or that have switched to a et al., 2006). The latter use of MβCD is attributed to its regenerative state sooner. Frontiers in Cellular Neuroscience | www.frontiersin.org 13 February 2019 | Volume 13 | Article 40 Roselló-Busquets et al. Cholesterol and Axonal Regeneration Our results indicate that lipid rafts are disrupted from the myelin-associated inhibitory agents. On the other, the beneficial cell membrane after MβCD treatment, as shown by the loss of effect might be due to an alteration of membrane ganglioside ratio CTxB staining. CTxB has affinity for the GM1 ganglioside, a or because lipid raft disruption promotes an environment that is member of the complex ganglioside family, which also includes favorable for neuron regeneration. GD1 and GT1 (Schnaar et al., 2014), and it has been widely In conclusion, we have found that lipid raft disruption used as a marker of lipid raft integrity (Kalka et al., 2001). It increases axonal growth in young neurons in vitro, enhances has been reported that treatment with MβCD destabilizes the axonal regeneration in two in vitro models, and promotes link between the axon and myelin as a result of the reduction peripheral nerve regeneration. Our results support the notion of GD1 and GT1 in the membrane. These two gangliosides that these membrane microdomains play an important role in act as a myelin-associated glycoprotein receptors (Vyas et al., the signaling responsible for determining neural growth during 2002) and, consequently, they mediate the inhibition of neurite development, as well as for nerve degeneration and regeneration outgrowth. With lipid raft disruption by MβCD, gangliosides are after injury. This could be important because many pathways removed from the cell membrane and they cannot mediate the and molecules are involved in the processes that occur after inhibition of neurite outgrowth. This situation may confer the nerve injury, and the environment plays a key role in the axon with a greater capacity to regenerate. Thus, the increased success or failure of nerve regeneration and functional recovery. motor and sensory reinnervation we observed after axotomy and However, caution should be taken before the translationability treatment with MβCD is consistent with a reduction of GD1 of our results. Considering the slow rate of nerve regeneration, and GT1 inhibition. in humans with considerable nerve length, target reinnervation Moreover, other studies have reported that cholesterol may need several months to year. The possible effects of removal affects various modes of endocytosis (Rodrigues et al., long-term lipid raft disruption treatment pointed above need 2013). Endocytosis is a widely used mechanism for receptor to be investigated in preclinical models before any potential internalization from the cell membrane, and it has been clinical application. demonstrated that cholesterol and lipid rafts play key roles in clathrin-independent endocytic (Lamaze et al., 2001) and in clathrin-dependent endocytic mechanisms (Rodal et al., 1999). AUTHOR CONTRIBUTIONS Thus, with lipid raft alteration, endocytosis might be impaired, thereby also remodeling the normal presence or removal of CR-B, RM-M, and MH-L designed, conducted, and analyzed different receptors at the cell surface. With cholesterol depletion the in vitro experiments. NO, JV, and XN designed, conducted, by MβCD, the endocytosis of neurotrophic factor receptors may and analyzed the in vivo experiments. CR-B, RM-M, MH-L, be inhibited (Hausott et al., 2011). As the improvement in motor and NO wrote the first draft of the manuscript. MP performed and sensory regeneration suggests, this inhibition would lead to the organotypic experiments. AM did percoll gradients to increased cell receptor availability at cell surface, thus facilitating isolated EGL neurons. RM-M, JV, XN, and ES designed axon regeneration. On the other hand, MβCD treatment might the experiments, analyzed the data, and wrote the final interfere with myelin as it has a high lipid and cholesterol content. version manuscript. Indeed, deficient cholesterol biosynthesis in oligodendrocytes delays myelination (Saher et al., 2005) and the maximal brain cholesterol synthesis corresponds to the peak of myelination FUNDING process (Korade and Kenworthy, 2008). However, our results Research in our laboratories was supported by grants from the indicate that MβCD treatment did not affect myelination of Spanish MINECO (SAF2016-76340R), CIBERNED, Spanish the regenerated nerve fibers in the adult mice. Nevertheless, MECD (FPU14/02156 and BES-2014-067857), TERCEL a detailed histomorphometric quantification of the regenerated (RD12/0019/0011) and ERDF funds. It was also supported axons might be needed for a quantitative evaluation of the degree by the European Union FPT-ICT projects NEBIAS (contract of remyelination after lipid raft disruption. number FP7-611687), EPIONE (FP7-602547), and FP7-NMP Finally, our results indicate that MβCD treatment accelerates MERIDIAN 280778. nerve regeneration after complete nerve lesion. The positive effect was detected as faster and higher levels of reinnervation of distal targets, such as plantar muscles, which are the most SUPPLEMENTARY MATERIAL discriminative for detecting differences in nerve regeneration rate. Whether these results would be more pronounced after a The Supplementary Material for this article can be found online more severe nerve injury requires further attention. at: https://www.frontiersin.org/articles/10.3389/fncel.2019. The results of this study shed light on the role of lipid 00040/full#supplementary-material rafts in peripheral nerve regeneration, although more research is needed to elucidate the specific mechanisms affected by lipid FIGURE S1 | Electrophysiological, locomotion and algesimetry tests performed after vehicle or MβCD treatment in uninjured mice. (A–D) Electrophysiological raft disruption. As this study was performed from a functional results in mice. Results of CMAP amplitude (A,B) and latency (C,D) recorded in perspective, a number of alterations may have been occurring. TA and PL muscles at 7, 14, 21, and 28 days after beginning of treatment. (E) Plot On the one hand, we may have accelerated regeneration due of the SFI obtained in walking track test. (F) Algesimetry test results during the to an increase in myelin clearance and thus a decrease in 4 weeks of treatment. 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Lipid rafts mediate the interaction between myelin-associated Pascual, Muhaisen, Navarro, del Valle and Soriano. This is an open-access article glycoprotein (MAG) on myelin and MAG-receptors on neurons. Mol. Cell. distributed under the terms of the Creative Commons Attribution License (CC BY). Neurosci. 22, 344–352. doi: 10.1016/S1044-7431(02)00031-3 The use, distribution or reproduction in other forums is permitted, provided the Vyas, A. A., Patel, H. V., Fromholt, S. E., Heffer-Lauc, M., Vyas, K. A., Dang, J., original author(s) and the copyright owner(s) are credited and that the original et al. (2002). Gangliosides are functional nerve cell ligands for myelin-associated publication in this journal is cited, in accordance with accepted academic practice. glycoprotein (MAG), an inhibitor of nerve regeneration. Proc. Natl. Acad. Sci. No use, distribution or reproduction is permitted which does not comply with these U.S.A. 99, 8412–8417. doi: 10.1073/pnas.072211699 terms. Frontiers in Cellular Neuroscience | www.frontiersin.org 16 February 2019 | Volume 13 | Article 40