Schwann cells use TAM receptor-mediated PNAS PLUS phagocytosis in addition to autophagy to clear myelin in a mouse model of nerve injury

Amanda Brosius Lutza,1, Won-Suk Chungb, Steven A. Sloanc, Glenn A. Carsonc, Lu Zhouc, Emilie Lovelettc, Sean Posadac, J. Bradley Zucherod, and Ben A. Barresc,1

aDepartment of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5125; bDepartment of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, South Korea; cDepartment of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305-5125; and dDepartment of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305-5125

Contributed by Ben A. Barres, July 24, 2017 (sent for review June 14, 2017; reviewed by Marc Freeman and Sarah Kucenas) Ineffective myelin debris clearance is a major factor contributing to molecular mechanisms that underlie it remain incompletely un- the poor regenerative ability of the central nervous system. In stark derstood (6–8). The postinjury scenario in the peripheral nerve is contrast, rapid clearance of myelin debris from the injured peripheral in stark contrast to that of the injured CNS, where myelin debris nervous system (PNS) is one of the keys to this system’s remarkable persists for months to even years after injury and is a major mo- regenerative capacity, but the molecular mechanisms driving PNS lecular roadblock to brain and spinal cord repair (9, 10). Given this myelin clearance are incompletely understood. We set out to dis- contrast, we reasoned that furthering mechanistic understanding of cover new pathways of PNS myelin clearance to identify novel strat- peripheral myelin clearance would not only answer important egies for activating myelin clearance in the injured central nervous outstanding questions in peripheral nerve biology, but may also system, where myelin debris is not cleared efficiently. Here we show that Schwann cells, the myelinating glia of the PNS, collaborate with provide insight into ways to promote myelin debris clearance in the hematogenous macrophages to clear myelin debris using TAM injured CNS. (Tyro3, Axl, Mer) receptor-mediated phagocytosis as well as auto- Myelin debris clearance in the injured peripheral nerve is a col- phagy. In a mouse model of PNS nerve crush injury, Schwann cells laborative effort accomplished by multiple cell types. Our laboratory NEUROSCIENCE up-regulate TAM phagocytic receptors Axl and Mertk following PNS and others have shown that macrophages recruited into the nerve injury, and Schwann cells lacking both of these phagocytic receptors from peripheral circulation use complement- and Fc receptor- exhibit significantly impaired myelin phagocytosis both in vitro and mediated mechanisms to help accomplish peripheral nervous sys- in vivo. Autophagy-deficient Schwann cells also display reductions in tem (PNS) myelin clearance. Accordingly, the abrogation of either myelin clearance after mouse nerve crush injury, as has been recently of these pathways in macrophages leads to decreases in macrophage shown following nerve transection. These findings add a mechanism, phagocytosis and delays in myelin clearance as well as impaired Axl/Mertk-mediated myelin clearance, to the repertoire of cellular regeneration and functional recovery (7, 10). Two types of tissue- machinery used to clear myelin in the injured PNS. Given recent resident glial cells have also been recognized to contribute to myelin evidence that astrocytes express Axl and Mertk and have previously debris removal from the injured peripheral nerve: Schwann cells and unrecognized phagocytic potential, this pathway may be a promising perineurial cells (11–13). Despite recent progress toward a molec- avenue for activating myelin clearance after CNS injury. ular understanding of the Schwann cell response to injury, our un- derstanding of the Schwann cell-mediated mechanism of myelin myelin | phagocytosis | Wallerian degeneration | Schwann cell | regeneration removal has remained incomplete (14, 15).

Significance ight regulation of debris clearance is an essential and highly Tconserved process required for successful development as well as maintenance of homeostasis and immune tolerance in Myelin is a potent inhibitor of axon regeneration. In the central mature tissues (1). Defects in clearance of debris are associated nervous system, failure to clear myelin debris after injury presents with diverse pathologies including , chronic a major roadblock to recovery. In contrast, rapid myelin clearance obstructive pulmonary disease and asthma, atherosclerosis, and in the peripheral nervous system (PNS) contributes to this system’s Alzheimer’s disease (2–4). In addition to being required in de- remarkable regenerative capacity, but the mechanisms involved veloping and healthy tissues, efficient debris clearance is essen- have remained incompletely understood. In this work, we set out tial for successful tissue repair following injury, when large to identify novel mechanisms of PNS myelin clearance to generate quantities of cellular debris accumulate over a short time span. new ideas about activating myelin clearance in the injured CNS. The cell types responsible for debris clearance include pro- We provide evidence that Schwann cells, myelinating glia of the fessional phagocytes recruited from the circulation, such as PNS, engulf myelin debris using two receptors, Axl and Mertk. We macrophages, as well as less renowned tissue-resident phago- hypothesize that astrocytes have the potential to use this same cytes. Elucidating the molecular mechanisms used by cells to mechanism to engulf myelin debris after CNS injury. clear debris during health and after injury is a critical step toward understanding how debris clearance goes awry in disease. Author contributions: A.B.L. and B.A.B. designed research; A.B.L., S.A.S., G.A.C., E.L., S.P., and L.Z. performed research; W.-S.C. contributed new reagents/analytic tools; A.B.L., A remarkable example of efficient debris clearance following S.A.S., G.A.C., E.L., J.B.Z., and B.A.B. analyzed data; and A.B.L. and B.A.B. wrote the paper. injury is removal of myelin debris from the lesioned peripheral Reviewers: M.F., Oregon Health & Science University; and S.K., University of Virginia. nerve. Following peripheral nerve injury, the nerve distal to injury The authors declare no conflict of interest. degenerates through the process of Wallerian degeneration, pro- Freely available online through the PNAS open access option. ducing a large quantity of myelin debris. Within only 2 to 3 wk after 1To whom correspondence may be addressed. Email: [email protected] or barres@ injury, the majority of this debris is cleared from the distal nerve stanford.edu. (5). This rapid clearance of peripheral myelin debris is essential for This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. successful axon regeneration and functional recovery, and yet the 1073/pnas.1710566114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1710566114 PNAS Early Edition | 1of9 Downloaded by guest on September 26, 2021 Here we report efforts to enhance our mechanistic un- didates using transgenic mice and in vitro and in vivo assays of derstanding of myelin removal by Schwann cells. To begin, we Schwann cell myelin clearance. examine the time course of Schwann cell and macrophage con- tributions to this process to establish the time window during Results which Schwann cell-mediated myelin clearance is at its peak. We Time Course of Schwann Cell-Mediated Myelin Clearance. We first then investigate two clearance mechanisms, autophagy and established the time frame of myelin clearance after PNS injury by phagocytosis, that might underlie Schwann cell clearance of my- measuring the quantity of residual myelin protein and compact elin. Next, using data gleaned from RNA sequencing (RNAseq) membranes in the mouse sciatic nerve distal to the site of nerve analysis of Schwann cells acutely purified from the intact and in- crush injury at multiple time points. We measured the myelin jured peripheral nerve, we identify candidate phagocytic pathways proteins myelin protein zero (MPZ or P0) and myelin basic pro- that might be necessary for this process. Finally, we test our can- tein (MBP) using Western blotting of distal nerve lysates (Fig.

Myelin clearance MBP A B Fluoromyelin Red C P0 0 2 4 7 10dpc 100 GAPDH --40 compact 80 membranes 60 MBP --18 40 MPZ --30 20 0 0246810

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Fig. 1. Schwann cells and macrophages contribute to myelin clearance after nerve crush. (A) Western blot depicting myelin protein clearance from the crushed peripheral nerve. Each lane represents a separate sciatic nerve, and each well was loaded with the same amount of total nerve protein. (B) Representative images depicting myelin compact membrane clearance from the sciatic nerve visualized using FluoroMyelin dye (red). (Scale bar, 20 μm.) (C) Quantification of myelin protein and compact membrane clearance. n = 3 nerves for each FluoroMyelin time point. n = 5 nerves for each MBP and MPZ time point. All data are presented as mean ± SEM. (D) Confocal single-z-plane images of intact and degenerating whole-mount sciatic nerves at 0, 2, 4, 6, and 9 dpc stained with p75 (Schwann cells; purple), Iba1 (monocytes/macrophages; green), MPZ (myelin; red), and DAPI (nuclei; blue). Arrows indicate myelin that appears to be “inside” Schwann cells. Arrowheads indicate macrophages that have engulfed myelin debris. (Scale bar, 20 μm.) (D′) Higher-magnification image of a phagocytic macrophage at 9 dpc and Schwann cell association with myelin at 6 dpc. (Scale bar, 10 μm.) (E) Oil red O accumulation in sciatic and splenic nerves degenerated in vitro for 7 d. DIV, days in vitro. (Scale bar, 20 μm.) (F and G) Time course of oil red O lipid droplet accumulation in Schwann cells at 0, 4, 9, and 17 d after injury (F) and macrophages at 9 d after injury (G). Cryosections were stained with S100 (Schwann cells; green) and Iba1 (macrophages; green). Lipid droplets are red (oil red O). Nuclei are blue (DAPI). Arrows indicate lipid droplets in Schwann cells. Arrowheads indicate lipid droplets in macrophages. (Scale bars, 10 μm.) (F′ and G′) Quantification of lipid droplet accumulation in Schwann cells and macrophages from 0 to 17 dpc. n = 3 nerves and 6 fields of view for each time point. All data are presented as mean ± SEM. (H) Time course of lysosome accumulation in the sciatic nerve at 0, 3, 5, and 7 dpc. Whole-mount sciatic nerves are stained with p75 (Schwann cells; purple), MBP (myelin; red), LAMP2 (lysosomes; green), and DAPI (nuclei; blue). Dashed lines outline myelin ovoids. Arrows indicate myelin that appears to be inside Schwann cells. (Scale bar, 10 μm.) *P < 0.05, **P < 0.01, ***P < 0.001.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1710566114 Brosius Lutz et al. Downloaded by guest on September 26, 2021 1A). Compact myelin membranes were measured by staining LC3 to LC3-PE at multiple time points using Western blotting, PNAS PLUS cryosections of the distal nerve with the lipophilic dye Fluo- and found that this measure increased modestly after nerve roMyelin red (Fig. 1B). In agreement with previously published crush, peaking at 4 d after injury (Fig. 2A). findings from our laboratory and others, we found that myelin We next used mice expressing the GFP fusion protein LC3-GFP clearance is already under way in the mouse peripheral nerve by to determine what cell type was up-regulating autophagy and the 2 d after injury and is ∼70 to 80% complete by 8 to 10 d after frequency of autophagosome formation in the injured nerve. The injury (Fig. 1C). LC3-GFP fusion protein allows the visualization of autophago- To elucidate the timing of individual cell-type contributions to somes when LC3-PE associates with the autophagosome mem- peripheral myelin clearance, we assessed the myelin clearance brane, but is diffusely distributed throughout the cytoplasm in the activity of monocytes/macrophages and Schwann cells at 2, 4, 6, absence of autophagy (19). We crossed these LC3-GFP mice to a and 9 d after sciatic nerve crush. We first used immunohisto- line of mice expressing cytoplasmic tdtomato in Schwann cells chemistry (IHC) for MPZ in combination with macrophage/ (loxSTOPlox tdtomato × P0 Cre) to obtain mice with green monocyte marker Iba1 and Schwann cell marker p75 to visualize autophagosomes and red Schwann cells. Examination of whole- clearance of myelin debris by both cell types. Throughout our mount sciatic nerves from these mice at 2, 4, and 7 d after injury immunohistochemical studies, Schwann cells were differentiated revealed that autophagosome formation occurs in Schwann cells from perineurial cells, which are also p75- and S100-immunoreactive, after sciatic nerve crush and reaches a maximum at 4 dpc, in by their location within the nerve, elongated cellular morphology, agreement with the results of our Western blotting experiment and characteristic association with axons and myelin (16). We (Fig. 2 B and C). Abundance of autophagosomes was highly var- interpreted the presence of myelin proteins inside of cells stained iable, however, with most cells exhibiting only very sparse or for Iba1 as evidence for myelin degradation by monocytes/macro- no autophagosomes. phages. Monocyte/macrophage degradation of myelin debris was To test whether autophagy is necessary for Schwann cell deg- apparent beginning at 6 d after injury and was even more pro- radation of myelin after nerve crush injury, as has recently been nounced at 9 d after injury (Fig. 1D, arrowheads and Fig. 1D′, Top). shown after nerve transection, we generated mice in which In contrast, we found that immunostaining for p75 and myelin Schwann cells are unable to perform autophagy due to deletion of protein was insufficient to deduce Schwann cell-mediated degra- essential autophagy protein atg7 (floxed Atg7 × P0 Cre) (20–22). dation of myelin. This difficulty arose from the fact that the intact These mice did not display any obvious behavioral abnormalities myelin sheath is already intimately associated with Schwann cell before their use for experimentsat8to12wkofage.Western

cytoplasm in the uninjured nerve, and from the fact that physical blotting of sciatic nerve Schwann cells purified from these animals NEUROSCIENCE fragmentation of the myelin sheath by Schwann cells ∼2dafter by immunopanning at 5 dpc indicated that conversion of LC3 to nerve injury to form myelin ovoids results in large amounts of LC3-PEhadbeeninhibitedasexpected(Fig.2D). Western blot segmented myelin that appears within the Schwann cell but has not analysis of residual peripheral myelin proteins MPZ and MBP from + − necessarily begun to be cleared or degraded by the cell (Fig. 1D, these autophagy-deficient Atg7flox/flox;P0 Cre / and littermate − − arrows and Fig. 1D′, Bottom). control Atg7flox/flox;P0 Cre / nerves indicated a significant re- To specifically detect Schwann cell degradation of myelin, duction in myelin clearance in sciatic nerves with autophagy- we turned to the lipid dye oil red O (ORO), which stains deficient Schwann cells at 7 d after crush injury. By 9 d after droplets of neutral lipids that arise following degradation of injury, this difference was no longer significant. The results of these the polar phospholipids of cell membranes (17). As expected, experiments led us to conclude that autophagy contributes to ORO brightly stains lipid droplets and only dimly stains intact Schwann cell-mediated clearance of myelin debris in mice after myelin and myelin ovoids, allowing us to specifically identify and sciatic nerve crush. Autophagy appears insufficient to fully account quantify lipid degradation (Fig. 1F). In our system, the ORO for Schwann cell-mediated myelin degradation, however, given the signal is specific to myelin degradation, since it was nearly absent paucity of autophagosomes observed in the postcrush nerve, in from mostly nonmyelinated degenerating splenic nerves (Fig. contrast with the abundance of ORO-positive lipid droplets within 1E). A time course of ORO-positive droplet formation revealed Schwann cells at 6 and 9 dpc. that myelin lipid degradation products accumulate in Schwann cells beginning at 4 d after injury, peak around 9 d after injury, Assessment of the Role of Schwann Cell Phagocytosis in Clearing and subside significantly by 2 wk after injury (Fig. 1F′), allowing Myelin Debris. We wanted to take an unbiased look at alternate us to conclude that Schwann cells contribute most heavily to mechanisms Schwann cells might use to clear myelin. To begin myelin clearance during this time window. ORO accumulation in this search, we used an RNAseq database recently generated by macrophages followed a very similar time course, also peaking at our laboratory that compares the transcriptomes of Schwann 9 days post crush (dpc), consistent with our IHC observations cells acutely purified from the intact and crushed rat sciatic nerve (Fig. 1 G and G′). As further evidence for degradative activity, at 0, 3, 5, and 7 dpc. DAVID software analysis of our lists Schwann cell lysosome abundance, measured by immunohisto- enabled us to take a first look at pathways enriched in Schwann chemistry using antibodies to LAMP2, also increased over the cells after nerve injury (23, 24). Notably, the first week after nerve injury (Fig. 1H). term “endosome” was significantly enriched in our gene lists in Lysosomes are interlinked with two main intracellular pro- comparison with the rat reference genome and enriched at in- cesses: endocytosis/phagocytosis and autophagy. We designed creasing levels of significance over time after injury, with P values experiments to determine whether these mechanisms mediate of 0.08 in the uncrushed nerve, 0.003 at 3 dpc, 0.007 at 5 dpc, and Schwann cell clearance of myelin. Our first set of experiments 0.001 at 7 dpc, suggesting that Schwann cells might use phago- aimed to assess whether Schwann cells require autophagy to cytosis in addition to autophagy to clear myelin debris. In- degrade myelin after peripheral nerve crush injury. deed, we found by immunohistochemistry using antibodies to endosome-specific protein EEA1 that endosomes are very Assessment of the Role of Autophagy in Myelin Debris Clearance. abundant in Schwann cells after nerve crush injury (Fig. 3A). To Lipidation of cytosolic mammalian myosin light chain 3 (LC3) to our knowledge, there is no existing transgenic mouse that is become LC3-phosphatidylethanolamine (LC3-PE) results in globally deficient in phagocytosis. Therefore, to assess the ne- transient association of this protein with nascent autophago- cessity of phagocytosis for Schwann cell-mediated myelin clear- somes and is critical for autophagosome formation (18). We ance, we sought to identify candidate molecular pathways that established a time course of autophagosome formation after we could then manipulate. We mined our RNAseq datasets for peripheral nerve crush by examining the ratio of unlipidated transcripts of known phagocytic receptors to identify specific

Brosius Lutz et al. PNAS Early Edition | 3of9 Downloaded by guest on September 26, 2021 pathways that might underlie Schwann cell phagocytosis of my- 600 A * elin debris. We selected candidate phagocytic receptors based on UC 4dpc two criteria: up-regulation by twofold or more after nerve injury, 400 GAPDH --40 and maximum fragments per kilobase million (FPKM) of at least 10. Two phagocytic receptors met both of these criteria: Axl and LC3-I --17 200 Mertk, both members of the TAM family of receptor tyrosine LC3-II --15 kinases with known roles in debris clearance in other systems 0 0246810(Fig. S1 and Fig. 3B) (3, 25). Interestingly, Megf10 and LRP1, Days post Crush both phagocytic receptors with known phagocytic functions in LC3II/LC3I (% of control) < 0 dpc other types of glia, were expressed at FPKM 6 (25, 26). We B C confirmed up-regulation of these receptors in the injured mouse 4 dpc LC3-GFP sciatic nerve at the protein level by Western blotting and spe- cifically in Schwann cells by using immunostaining (Fig. 3 C D LC3-GFP and ). Are Axl and Mertk required for Schwann cell phagocytosis of

LC3-GFP / TdT DAPI myelin? We first assessed this question in primary culture. We 2 dpc began by purifying myelin from peripheral nerves and labeling it with the pH-sensitive dye pHRODO, which fluoresces brightly in acidic cellular compartments such as lysosomes but exhibits only dim fluorescence in the cytosol. When purified wild-type Schwann cells isolated from injured sciatic nerves were fed pHRODO- labeled myelin, the cells readily engulfed the labeled debris and 4 dpc LC3-GFP/TdTomato/DAPI exhibited pHRODO fluorescence. Although outside the realm of this study, Schwann cells were also observed to cluster over several hours of incubation with myelin debris, suggesting migra- tion toward debris over time. We could quantify the level of pHRODO fluorescence per cell using flow cytometry (FACS) analysis following trypsinization of the Schwann cells or per A–C 7 dpc field using live-cell microscopy (Fig. 4 ). We performed our FACS-based in vitro phagocytosis assay using Schwann cells purified from the sciatic nerves of WT mice and mice lacking Axl or Mertk. The results of these experiments revealed that in comparison with Schwann cells purified from wild-type litter- mate controls, Schwann cells lacking both copies of Axl or Mertk exhibited significant 40–50% defects in myelin phagocytosis (Fig. D Atg7 flox/flox E MBP 4 D and E). Based on these findings, we were very interested 100 P0Cre -/- P0 Cre +/- Control in knowing if Schwann cells lacking both Axl and Mertk would GAPDH --40 80 cKO exhibit an even greater defect in phagocytic ability. Indeed, 60 double-mutant Schwann cells were almost completely unable to --17 n.s. F LC3-I 40 phagocytose myelin debris (Fig. 4 ). We confirmed these data using LC3-II --15 ** 20 n.s. live-cell microscopy of Axl/Mertk WT, double-heterozygous (DHet), and double-mutant (DKO) Schwann cells over a 24-h

Atg7 --80 Myelin (% of control) 0 5 7 9 period of coincubation with pHRODO-labeled myelin debris (Fig. F UC Days post crush 4G). Interestingly, this experiment revealed a significant defect in the phagocytic ability of Axl/Mertk DHet Schwann cells relative to MPZ WT controls, suggesting haploinsufficiency of these receptors, at 100 Control cKO least in the in vitro setting. 80 n.s. We next tested the necessity of the Axl and Mertk pathways for n.s. 60 ** myelin clearance after peripheral nerve injury in vivo. We quan- 40 tified residual myelin proteins MPZ and MBP 7 and 9 d after crush − − − − + − + − 20 in sciatic nerves from Axl / ;Mertk / (DKO) and Axl / ;Mertk /

Myelin (% of control) 0 (DHet) littermates as well as wild-type controls. As observed in our 5 7 9 UC autophagy experiments, we found a significant decrease in myelin Days post crush protein clearance in nerves lacking both Axl and Mertk at 7 dpc in Fig. 2. Schwann cell autophagy contributes to myelin clearance after nerve comparison with Axl/Mertk WT nerves (Fig. 5 A–C). To further crush. (A) Western blot of whole-sciatic nerve protein lysate at 0 (UC) and 4 dpc characterize this phenotype, we examined myelin morphology in and graph of LC3II/LC3I values for the first 10 d post injury. Each Western blot WT and DKO nerves at 7 dpc using electron microscopy (EM), lane represents a separate sciatic nerve, and each well was loaded with the reasoning that if Axl/Mertk mutant Schwann cells were unable to same amount of total nerve protein. All data are presented as mean ± SEM. engulf myelin debris, myelin in these mutant nerves would persist n = 3 nerves for each time point. (B) Representative confocal single-z-plane + − + − in a more preserved state than in WT injured nerves. By EM, images of whole-mount sciatic nerves from LC3-GFP / ;P0 Cre / ;loxSTOPlox + tdtomato flox/ mice at 0, 2, 4, and 7 dpc. Autophagosomes are green (LC3-GFP), Schwann cells are red (tdtomato), and nuclei are blue (DAPI). (Scale bar, 50 μm.) + − (C) Maximum-intensity projection from a stack of confocal images of tissue MBP and MPZ in sciatic nerves from Atg7 flox/flox P0 Cre / mice. Protein at 4 dpc. Arrows denote autophagosomes in a Schwann cell. (Scale bar, lysates were generated from single nerves, and each Western blot well was 20 μm.) (D) Western blot of immunopanned Schwann cells purified 5 d after loaded with the same amount of total nerve protein. n = 3 or 4 nerves per + − − − + − crush from Atg7 flox/flox;P0 Cre / and Atg7 flox/flox;P0 Cre / mice. Each time point per genotype. cKO, Atg7 flox/flox,P0 Cre / ; control, Atg7 flox/ lysate was prepared from all of the Schwann cells purified from two sciatic flox;P0 Cre−/−. Data are presented as mean ± SEM. n.s., not significant; *P < nerves. (E and F) Quantification of Western blots of residual myelin proteins 0.05, **P < 0.01.

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1710566114 Brosius Lutz et al. Downloaded by guest on September 26, 2021 A an intracellular vacuole, appeared more abundant in DKO nerves PNAS PLUS in comparison with WT nerves (Fig. S2A). This phenotype was statistically significant when quantified using toluidine blue-stained thick sections by a blinded observer (Fig. S2 B and C). Without 3D reconstruction EM, we were unable to determine with certainty whether these preserved myelin figures were extracellular or as- sociated with a Schwann cell nucleus out of the plane of section. These data corroborated our Western blot findings and provided

EEA1 / P75 DAPI additional evidence for a delay in degradation of myelin in Axl/ Mertk double-mutant nerves. To confirm that reduced myelin clearance in Axl/Mertk DKO nerves was Schwann cell-mediated, we compared the number of oil red O droplets as well as the number of endosomes in Axl/ B Mertk Mertk WT, double-heterozygous, and double-knockout Schwann 60 Axl cells post injury (Fig. 5 E–G). We measured endosome abundance Megf10 using an antibody to EEA1, an endosome-specific protein. LRP1 Schwann cells in Axl/Mertk double-knockout nerves exhibited 40 reduced numbers of both oil red O droplets as well as endosomes in comparison with the Schwann cells of WT controls. In our

FPKM endosome experiment, we again saw evidence for potential hap- 20 loinsufficiency of Axl and Mertk, this time in vivo, as endosome number was also significantly reduced in DHet Schwann cells relative to WT controls. These experiments demonstrated that 0 02468 Schwann cells use Axl and Mertk to clear myelin debris in vivo. days post crush Discussion C 0 2 4 7 10 dpc The Peak Periods of Schwann Cell- and Macrophage-Mediated Myelin

Clearance Are More Coincident than Previously Believed. Previously, NEUROSCIENCE --200 Mertk myelin clearance in the injured peripheral nerve had been --150 thought to occur in two stages: an initial Schwann cell-mediated period of clearance 0 to 6 d after injury, followed by a second, Axl --120 macrophage-mediated period of clearance. This picture of my- elin clearance is based on studies by our laboratory and others demonstrating that knockdown of specific pathways involved in D 0 dpc 5 dpc macrophage-mediated clearance results in a delay in myelin clearance, but only from day 5–6 post injury onward (6, 7). Here we show that although Schwann cells partition their myelin sheath to form myelin ovoids during the first 2–3 d after injury,

Mertk / tdt DAPI intracellular generation of lipid droplets in Schwann cell (SC) $$ cytoplasm follows a very similar time course as the appearance of lipid droplets in macrophages, beginning at ∼4 d after injury and peaking at 9 d post injury (Fig. 1).

/ tdt DAPI Schwann Cells Use Autophagy to Clear Myelin Debris. Descriptive Axl studies have long postulated the involvement of autophagy and/ Fig. 3. Schwann cells up-regulate the phagocytic receptors Axl and Mertk or phagocytosis in myelin clearance by Schwann cells (28, 29). after nerve crush. (A) Representative IHC image of a sciatic nerve 9 dpc Indeed, the intimate association of myelin with Schwann cell stained for endosomes (EEA1; green), Schwann cells (p75; red), and nuclei cytoplasm both before and after injury makes autophagy an attractive μ (DAPI; blue). (Scale bar, 10 m.) Arrows indicate regions of colocalization of mechanism for myelin clearance. Alternatively, the involvement EEA1 and p75 immunoreactivity. (B) Graphical illustration of the time course of expression of select phagocytic in acutely purified rat Schwann cells of macrophages in myelin phagocytosis implies accessibility of at 0, 3, 5, and 7 dpc. The dotted line represents an average FPKM of 11 for myelin debris to phagocytic cell types, making phagocytosis by the dataset. FPKM values are averaged across two samples for each time Schwann cells a plausible mechanism for clearance as well. point. (C) Western blot showing up-regulation of Mertk and Axl protein Here we have provided evidence that both of these processes after mouse sciatic nerve crush. Protein lysates were generated from single contribute to Schwann cell-mediated myelin clearance. Gomez- nerves, and each Western blot well was loaded with the same amount Sanchez et al. recently demonstrated that Schwann cells use + of total nerve protein. (D) Cross-sections of loxSTOPlox tdtomato flox/ ;P0 autophagy to degrade myelin after nerve transection, a process +/− Cre mouse sciatic nerve at 0 and 5 dpc stained by IHC with antibodies to they coined “myelinophagy” (22). These findings were supported Mertk and Axl (green) and DAPI. Arrows highlight Schwann cells in each in a subsequent publication by Jang et al. (20). Given the known field. (Scale bar, 10 μm.) association between nutrient restriction and autophagy and the more drastic disruption in distal nerve blood supply caused by myelin in both WT and DKO nerves existed at various stages of transection injury in comparison with crush injury, we wondered if these findings would apply in a nerve crush scenario as well degradation at 7 dpc, with highly degraded myelin figures localized (30). Our data corroborate these studies showing the involvement within vacuoles of Schwann cells and macrophages, consistent with of autophagy in Schwann cell-mediated myelin clearance and previous ultrastructural studies of Wallerian degeneration (27). validate this mechanism in a crush model of nerve injury (Fig. 2). Upon close examination, we noted that preserved, nonvacuolar However, our findings also indicate that autophagy alone does myelin figures, each defined as a single ring of closely opposed not account for all of the myelin clearance activity exhibited by myelin membranes (no onion bulb appearance) not located within Schwann cells after nerve crush.

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Fig. 4. Schwann cells use Axl and Mertk to clear myelin debris in vitro. (A) Schematic illustration of a Schwann cell in vitro myelin phagocytosis assay. (B) Representative FACS tracing of Schwann cells analyzed by flow cytometry after no exposure and 2-, 4-, and 8-h exposure to pHRODO-labeled myelin. (C) Representative live-cell images of Schwann cells before and 8 h following addition of pHRODO-labeled myelin to culture media. (Scale bar, 50 μm.) (D–F) Results of an in vitro phagocytosis assay performed on Schwann cells purified from Axl−/−,Mertk−/−, and Axl/Mertk double-mutant Schwann cells and their lit- termate controls. Cells were purified from sciatic nerves 6 d after crush. Myelin phagocytosis was quantified using flow cytometry 2 to 3 h after addition of pHRODO-labeled PNS myelin debris. n = 4 for each genotype. Data are presented as mean ± SEM. (G) Quantification of integrated fluorescence per live-cell imaging field of Schwann cells coincubated with pHRODO-labeled myelin at 2 to 24 h after addition of myelin. n = 3 for each genotype: wild type, double heterozygote, and double knockout. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001.

Schwann Cells Help to Clear Myelin Debris by the Axl/Mertk Phagocytic recruited into the nerve after injury. We conducted preliminary Pathways. Using a Schwann cell RNAseq transcriptome to identify studies to test this hypothesis using clodronate liposomes to re- candidate phagocytic pathways involved in myelin clearance, we duce macrophage number (32). These studies were unable to test have shown that two phagocytic receptors, Axl and Mertk, are our prediction due to an inability to reduce macrophage number up-regulated by Schwann cells after injury and contribute to more than 50% using this method and an observed significant Schwann cell clearance of myelin in vitro and in vivo (Figs. 3–5). reduction in Schwann cell phagocytosis in wild-type clodronate- The TAM family of receptors is well-known for its involvement in treated animals versus untreated animals. The fact that clodronate phagocytosis by tissue-resident phagocytes: by retinal pigment ep- treatment, itself, led to reduced myelin clearance by Schwann cells ithelial cells in the retina, Sertoli cells in the testis, and astrocytes in raises the possibility that macrophages are required for Schwann the brain (25, 31). Our study adds a cell type, debris type, and cells to attain their full phagocytic potential. Indeed, our RNAseq tissue setting to this list and raises several issues for consideration. transcriptomes indicate that Gas6, one of the bridging molecules First, we show that both Schwann cell autophagy and Schwann required for TAM receptor function, is highly expressed by mac- cell phagocytosis contribute to myelin clearance after nerve injury. rophages isolated from the injured peripheral nerve. Other pos- In vivo, however, the impact of each of these mechanisms on the sible compensatory mechanisms include activation of alternate amount of myelin debris remaining in the injured nerve is con- Schwann cell pathways and increased clearance by yet other cell strained to a relatively short time window which begins around types such as perineurial cells (11). The redundancy of multiple a week after injury and ends by 9 d post injury (Fig. 5). It is very cellular and molecular mechanisms to mediate myelin clearance in likely that the role of these pathways in our mutant mice is the injured peripheral nerve highlights the complexity of this masked by compensatory increased phagocytosis by macrophages process and its importance for successful nerve repair. In future

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1710566114 Brosius Lutz et al. Downloaded by guest on September 26, 2021 A BCMPZ MBP PNAS PLUS 100 n.s. 100 GAPDH WT WT 80 DHet 80 n.s. DHet MPZ DKO DKO 60 ** 60 40 40 n.s. ** MBP 20 20 n.s. Myelin (% of control) 0 Myelin (% of control) 0 7dpc: CR UC CR UC CR UC 4 7 9 4 7 9 UC UC WT DHet DKO Days post crush Days post crush D WT DKO E 100 * * *** n.s. 80

60

WT DKO 40

20

EEA1 / P75 DAPI *

Endosomes per Schwann cell 0

WT DHet DKO NEUROSCIENCE

FGS100/ORO/DAPI WT

* 100 n.s. WT 80 *** DHet DKO 60 ** DKO 40

20 * 0 7 9 Lipid droplets per field in Schwann cells

Fig. 5. Schwann cells use Axl and Mertk to clear myelin debris in vivo. (A) Representative Western blot showing myelin protein (MBP and MPZ) levels in wild- type, Axl/Mertk double-heterozygous, and Axl/Mertk double-knockout mouse sciatic nerves at 0 and 7 dpc. Protein lysates were generated from single nerves, and each Western blot well was loaded with the same amount of total nerve protein. CR, crushed; UC, uncrushed. (B and C) Quantification of MPZ- and MBP- stained Western blots of protein lysates from WT, Axl/Mertk double-heterozygous, and Axl/Mertk double-knockout mouse sciatic nerves at 0, 4, 7, and 9 dpc. n = 3 to 10 per genotype per time point. Data are presented as mean ± SEM. (D) Representative IHC images of cryosections of 9-dpc WT and Axl/Mertk DKO mouse sciatic nerves stained with antibodies to EEA1 (endosomes) and p75 (Schwann cells). Arrowheads indicate colocalization of EEA1 and p75, while as- terisks indicate EEA1 immunoreactivity in a p75-negative cell, presumably a macrophage. (Scale bar, 10 μm.) (E) Quantification of IHC images of EEA1- and p75-labeled Schwann cells. Graph of average endosome abundance (EEA1 puncta) per Schwann cell at 9 dpc in Schwann cells from WT, Axl/Mertk double- heterozygous, and Axl/Mertk double-knockout mice. Eight cells were blindly selected and analyzed per animal. n = 4 or 5 animals per genotype. Data are presented as mean ± SEM. (F) Representative IHC images of 7-dpc cryosections of WT and Axl/Mertk DKO mouse sciatic nerves stained with ORO (lipid droplets) and antibodies to S100 (Schwann cells). Arrowheads indicate ORO within an S100-positive cell, while asterisks indicate ORO-positive droplets in S100- negative cells, presumably macrophages. (Scale bar, 10 μm.) (G) Quantification of ORO-positive lipid droplet abundance per field at 7 and 9 dpc in blindly selected and analyzed fields of IHC-stained tissue from WT, Axl/Mertk double-heterozygous, and Axl/Mertk double-knockout mice. n = 4 to 10 animals per genotype per time point. Four images were analyzed per animal. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

experiments, it would be interesting to observe the effect on my- A fascinating area of future study is understanding how myelin, elin clearance in a system where Schwann cells are unable to which is protected from attack by phagocytic cells in the healthy perform both phagocytosis and autophagy. Additionally, it may nerve, becomes a phagocytic target after injury. At a structural be the case that autophagy and/or TAM-mediated phagocytosis level, one possibility is that following myelin ovoid formation, contribute to regulating myelin turnover and maintenance in the myelinating Schwann cells shed their myelin into the extracellular healthy peripheral nerve, which has only a very sparse population space to be engulfed by debris-scavenging phagocytes. Another of resident macrophages. possibility is that phagocytes play an active role in promoting

Brosius Lutz et al. PNAS Early Edition | 7of9 Downloaded by guest on September 26, 2021 release of myelin from intact myelin sheaths by inducing local goat anti-P75NTR (Neuromics; 1:500), rabbit anti-Iba1 (Wako; 1:300), rabbit out-foldings in the myelin sheath akin to the myelinosome for- anti-EEA1 (Abcam; 1:200), chicken anti-MPZ (Millipore; 1:100), rat anti-LAMP2 mation recently reported by Romanelli et al. in a mouse model (Abcam; 1:100), goat anti-Axl (R&D Systems; 1:100), and goat anti-Mertk (R&D of multiple sclerosis (33). In this latter scenario, myelin may Systems; 1:100). Secondaries used were donkey or goat Alexa Fluor 488/ 594/568/405 (highly cross-absorbed; Invitrogen)-conjugated and used at never be apparent in the extracellular milieu, since it would be 1:1,000 on cryosections and at 1:500 for whole mounts. directly pinched off from the intact myelin sheath by phagocytic For staining of splenic and sciatic nerves degenerated in vitro, following processes. At a molecular level, “eat me” signals on the surface removal of epineurium and superfluous connective tissue, nerves were cut of myelin may be exposed or up-regulated after injury, leading to into 5-mm segments, transferred to DMEM supplemented with 5% FBS, and

interaction with phagocytic receptors and promoting engulfment, incubated at 37 °C/5% CO2 until fixation and staining as described above. as is observed in clearance of apoptotic cells (2). Finally, the specificity of Axl and Mertk receptors for myelin Western Blotting. On dry ice, nerves were ground using ReadyPrep Mini debris among the multiple kinds of cellular debris present in the Grinders (Bio-Rad) for 15 s. RIPA (200–400 μL) with cOmplete Ultra tablets postinjury nerve has yet to be thoroughly investigated. Schwann (Roche) was added, and samples were ground for another 30 s and then × cells may use these receptors to clear axonal as well as myelin centrifuged (16,000 g, 4 °C). For Western blots of whole-nerve lysates, pro- tein concentration was determined using a BCA Kit (Pierce), and equal debris. Alternatively, macrophages rather than Schwann cells amounts of total protein were loaded per well. For Western blots of immu- may clear axon debris after injury. Using our growing knowledge nopanned Schwann cell lysate, cells were lysed by addition of RIPA with of the molecular interactions that enable phagocytes to engage cOmplete Ultra tablets (Roche) and the entire lysate was loaded onto the gel. with targets, it will be very interesting to understand how specific Samples were run through an SDS/polyacrylamide gel and transferred onto cell types and molecular pathways are coordinated to accomplish PVDF membranes (Immobilon-FL 0.45 μm; Millipore). Blots were washed, dried debris clearance in the peripheral nerve (1, 2). (RT, overnight), rehydrated, blocked (Odyssey buffer; LI-COR), and incubated with relevant primary and secondary (IRDye 680LT/800CW; LI-COR) antibodies. Myelin Phagocytosis Pathways May Be Dysfunctional After CNS The following primary antibodies were used for Western blotting experi- Injury. Why CNS glia fail to clear myelin debris after CNS in- ments: rabbit anti-LC3 (Novus Biologicals; 1:1,000), rabbit anti-ATG7 (Cell jury is a critically important question. Persistent myelin debris in Signaling Technology; 1:750), chicken anti-GAPDH (ProSci; 1:1,000), mouse anti-MPZ (obtained as a gift from Juan J. Archelos, University of Graz, Graz, the injured CNS milieu is one of the major obstacles to successful Austria; 1:10,000), rat anti-MBP (Abcam; 1:1,000), goat anti-Axl (R&D Systems; CNS repair (34). Unlike Schwann cells, oligodendrocytes, the 1:1,000), and goat anti-Mertk (R&D Systems; 1:1,000). Western blots were la- myelinating glia of the CNS, do not express phagocytic ma- beled with IRDye 680LT/800CW secondary antibodies (LI-COR; 1:20,000). chinery and do not contribute to myelin debris clearance (22, 34, 35). Macrophages do not enter the distal segment of axotomized In Vitro Schwann Cell Phagocytosis Assay. Schwann cells were purified 6 d CNS axon pathways, as the blood–brain barrier does not break after sciatic nerve crush from 8- to 13-wk-old mice according to the methods down and microglia appear capable of only a limited degree of described in ref. 39. Cells were plated at equal density into wells of a 96-well myelin debris clearance (10, 36). Recent work from our labora- plate. The following day, cells in each well were rinsed once with Dulbecco’s tory, however, has shown that mouse astrocytes express phago- phosphate-buffered saline (DPBS) and fed media supplemented with 5% FCS and 1 μL pH-sensitive dye (pHRODO)–labeled crude PNS myelin purified cytic pathways and that they phagocytose synaptic debris in the according to ref. 37. Myelin was quantified using the BCA assay and used at developing and adult brain using Mertk, Megf10, and LRP1 and a protein concentration of 800 mg/mL. For flow cytometry analysis, Schwann cells

robustly phagocytose myelin debris in vitro (25, 37). Why then, were trypsinized after incubation at 37 °C/5% CO2 for 2–3 h and resuspended in are astrocytes unable to phagocytose myelin debris after CNS 30% FCS on ice. Data were collected at the Stanford Shared FACS Facility injury? Gene profiling of reactive astrocytes following CNS in- obtained using the facility’s LSR II.UV. SCs were identified by forward scatter, side jury demonstrates that many components of these phagocytic scatter, and viability (DAPI) gating. Mean PE Texas red fluorescence (pHRODO) pathways are down-regulated and that after CNS axotomy a type was calculated for each cell sample. For live-cell imaging, cells were incubated at of reactive astrocyte is generated that has little ability to phagocy- 37 °C/5% CO2 and each well was imaged at 15-minintervalsusinganIncuCyte tose (37, 38). As we continue to uncover the Schwann cell-mediated Zoom Live-Cell Analysis System (Essen BioScience). Integrated fluorescence in- tensity was quantified for each well. Images displaying no fluorescence due to mechanism of myelin debris clearance, it will be interesting to use evident error in automated focal plane were excluded from analysis. our findings to diagnose specific deficiencies in myelin clearance pathways after CNS injury and to devise therapeutic strategies for Mice. C57BL/6 mice were obtained from Charles River. Mertk and Axl mutant activating this process. mice were obtained from Greg Lemke at The Salk Institute, San Diego, CA. Atg7f/f mice were obtained from David Sulzer at Columbia University, New Materials and Methods York, NY, with consent from Masaaki Komatsu at the Tokyo Metropolitan Immunohistochemistry and Fluorescent Dye Labeling. Nerves were para- Institute of Medical Science, Tokyo, Japan. LC3-GFP mice were obtained from formaldehyde (PFA)-fixed for 4 h on ice, cryopreserved with 30% sucrose in PBS Thomas Rando at Stanford University with consent from Noboru Mizushima, overnight (4 °C), embedded in 2:1 OCT:30% sucrose, and sliced (10 to 12 μm) as RIKEN BRC, Ibaraki, Japan. P0 Cre and loxSTOPlox tdtomato mice were longitudinal or cross-sections. Slides were dried at 60 °C, placed in PBS, blocked obtained from JAX (stock nos. 017927 and 007914). Animals were housed for 1 h [room temperature (RT), in 10% serum/0.2% Triton X-100 in PBS], and and handled in accordance with the guidelines of the Administrative Panel incubated with primary antibodies overnight (4 °C, in 10% serum/0.2% Triton on Laboratory Animal Care of Stanford University. Unless noted otherwise, X-100 in PBS). They were then washed with PBS, given secondary antibodies all mice were 8–13 wk of age at the time of the experiment, and compar- for 1 h (RT, in 10% serum/0.2% Triton X-100 in PBS), washed in PBS, and isons between genotypes were made between matched littermate controls. coverslipped in Vectashield with DAPI (Vector Laboratories). For experiments involving Axl/Mertk mutant mice, double-knockout animals For sciatic nerve whole mounts, nerves were PFA-fixed for 4 h on ice. Each were matched with littermate double-heterozygous controls. WT controls nerve’s perineurium was carefully removed with forceps, permeabilized with for these experiments were age-matched only (to the closest week). For methanol on dry ice, and then blocked with 10% serum/1% Triton X-100/PBS electron microscopy experiments, double-knockout animals were age-matched overnight at 4 °C. Nerves were incubated with primary antibodies in 10% se- only (to the closest week) with WT controls. rum/1% Triton X-100/PBS for 48 h (4 °C), washed with PBS/1% Triton X-100, and then incubated with secondary antibodies in 10% serum/1% Triton X-100/PBS Statistical Analysis. All measurements in this study are presented as means ± for 48 h (4 °C). After incubation with secondary antibodies, nerves were washed SEM. Significance was determined with two-tailed unpaired Student’s t test, with 1% Triton X-100/PBS, put through 25%, 50%, and 75% glycerol in PBS and P < 0.05 was considered significant. *P < 0.05, **P < 0.01, ***P < 0.001. (>6 h each, 4 °C), and then mounted. Oil red O staining (Abcam; ab150678) For analysis of Axl/Mertk live-cell imaging data, a two-way ANOVA with was performed according to the manufacturer’s instructions. FluoroMyelin Tukey test was performed. staining was performed with FluoroMyelin Red Fluorescent Myelin Solution (Life Technologies) according to the manufacturer’s instructions. The follow- Sciatic Nerve Crush. All surgical experiments were performed under 2.5% ing antibodies were used for immunostaining: rabbit anti-S100 (Dako; 1:100), isoflurane. Sciatic nerve crush injury was performed as previously described

8of9 | www.pnas.org/cgi/doi/10.1073/pnas.1710566114 Brosius Lutz et al. Downloaded by guest on September 26, 2021 (40). Briefly, the sciatic nerve was exposed at midthigh level on the left side Epifluorescent Image Acquisition and Analysis. Epifluorescence images were PNAS PLUS of the animal and crushed with smooth forceps for 30 s. The upper thigh was taken with a Zeiss Axio Imager M1. Confocal images were taken with an shaved and sterilized using isopropanol. A 1-cm incision was made using a LSM 710 confocal microscope at the Stanford Neuroscience Microscopy scalpel, and the nerve was visualized via blunt dissection using forceps. The Service, supported by NIH NS069375. Live-cell images were taken using left sciatic nerve was crushed at midthigh for 10 s using forceps marked with the IncuCyte Zoom Live-Cell Analysis System (Essen BioScience). Image sterile graphite to mark the crush site. Carprofen (5 mg/kg s.c.) was ad- analysis was carried out using Fiji ImageJ and Image Studio. Western blots ministered for analgesia. were imaged with the Odyssey CLx Imaging System (LI-COR) in 700- and 800-nm channels. Electron Microscopy Image Acquisition and Analysis. Transmission electron microscopy of sciatic nerves was performed in conjunction with the Stanford ACKNOWLEDGMENTS. We thank members of the B.A.B. laboratory, notably Cell Sciences Imaging Facility supported by NIH 1S10RR02678001. Sciatic nerves Mariko Bennett and Anja Scholze, for discussions and support. We also were processed essentially as described for optic nerves (41). Briefly, mice were thank Greg Lemke for helpful comments on experimental design, and David killed by CO2 inhalation. Sciatic nerves were dissected out and postfixed in cold Parkinson and Xin-Peng Dun for advice on the whole-mount staining Karlsson–Schultz fixative (2.5% glutaraldehyde, 4% PFA in phosphate buffer, protocol. This work was supported by the Dr. Miriam and Sheldon G. Adelson pH 7.3) at 4 °C, treated with 2% osmium tetroxide in cold Karlsson–Schultz Medical Research Foundation (B.A.B.), Christopher and Dana Reeve fixative, serially dehydrated, and embedded in EMbed 812 (EMS; 14120); Foundation (B.A.B.), NIH Grants EY11310, NS069375, 1S10RR02678001, and 85-nm sections were transferred onto formvar/carbon-coated 50-mesh copper NIH S10 Shared Instrument Grant S10RR027431-01 (to B.A.B.), NIH Grant T32 HD007249 Developmental and Neonatal Biology Training Program (to grids and stained for 30 s in 3% uranyl acetate in 50% acetone followed by A.B.L.), National Institute of Mental Health Grants T32GM007365 and staining for 3 min in 0.2% lead citrate. Images were acquired with a JEOL F30MH106261, and Bio-X Predoctoral Fellowship (to S.A.S.), and the Stan- μ 1400 transmission electron microscope; 1- m sections were stained with tolu- ford Medical Scientist Training Program and School of Medicine (A.B.L.). idine blue and imaged with a Zeiss Axio Imager M1. For quantification of W.-S.C. is supported by National Research Foundation of Korea (NRF) preserved myelin figures in toluidine blue-stained sections, preserved myelin Grants NRF-2016M3C7A1905391 and NRF-2016R1C1B3006969 funded by figures were counted for two 40× images of each nerve. Analysis of toluidine the Korean government (MSIP). J.B.Z. is a Career Transition Award Fellow blue sections was blinded to genotype. of the National Multiple Sclerosis Society.

1. Arandjelovic S, Ravichandran KS (2015) Phagocytosis of apoptotic cells in homeostasis. 22. Gomez-Sanchez JA, et al. (2015) Schwann cell autophagy, myelinophagy, initiates Nat Immunol 16:907–917. myelin clearance from injured nerves. J Cell Biol 210:153–168. 2. Elliott MR, Ravichandran KS (2010) Clearance of apoptotic cells: Implications in health 23. Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of and disease. J Cell Biol 189:1059–1070. large gene lists using DAVID Bioinformatics Resources. Nature Protoc 4:44–57. 3. Burstyn-Cohen T, et al. (2012) Genetic dissection of TAM receptor-ligand interaction 24. Huang DW, Sherman BT, Lempicki RA (2009) Bioinformatics enrichment tools: Paths

– NEUROSCIENCE in retinal pigment epithelial cell phagocytosis. Neuron 76:1123 1132. toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37: 4. Hong S, et al. (2016) Complement and microglia mediate early synapse loss in Alz- 1–13. – heimer mouse models. Science 352:712 716. 25. Chung WS, et al. (2013) Astrocytes mediate synapse elimination through MEGF10 and 5. Gaudet AD, Popovich PG, Ramer MS (2011) Wallerian degeneration: Gaining perspective MERTK pathways. Nature 504:394–400. on inflammatory events after peripheral nerve injury. JNeuroinflammation8:110. 26. Ziegenfuss JS, et al. (2008) Draper-dependent glial phagocytic activity is mediated by 6. Vargas ME, Watanabe J, Singh SJ, Robinson WH, Barres BA (2010) Endogenous anti- Src and Syk family kinase signalling. Nature 453:935–939. bodies promote rapid myelin clearance and effective axon regeneration after nerve 27. Reichert F, Saada A, Rotshenker S (1994) Peripheral nerve injury induces Schwann cells injury. Proc Natl Acad Sci USA 107:11993–11998. to express two macrophage phenotypes: Phagocytosis and the galactose-specific 7. Dailey AT, Avellino AM, Benthem L, Silver J, Kliot M (1998) Complement depletion – reduces macrophage infiltration and activation during Wallerian degeneration and lectin MAC-2. J Neurosci 14:3231 3245. axonal regeneration. J Neurosci 18:6713–6722. 28. Satinsky D, Pepe FA, Liu CN (1964) The neurolemma cells in peripheral nerve de- – 8. Brown MC, Perry VH, Hunt SP, Lapper SR (1994) Further studies on motor and sensory generation and regeneration. Exp Neurol 9:441 451. nerve regeneration in mice with delayed Wallerian degeneration. Eur J Neurosci 6:420–428. 29. Holtzman E, Novikoff AB (1965) Lysosomes in the rat sciatic nerve following crush. 9. Schwab ME (2004) Nogo and axon regeneration. Curr Opin Neurobiol 14:118–124. J Cell Biol 27:651–669. 10. Vargas ME, Barres BA (2007) Why is Wallerian degeneration in the CNS so slow? Annu 30. Ravikumar B, et al. (2010) Regulation of mammalian autophagy in physiology and Rev Neurosci 30:153–179. pathophysiology. Physiol Rev 90:1383–1435. 11. Lewis GM, Kucenas S (2014) Perineurial glia are essential for motor axon regrowth 31. Lemke G (2013) Biology of the TAM receptors. Cold Spring Harb Perspect Biol 5: following nerve injury. J Neurosci 34:12762–12777. a009076. 12. Liu HM, Yang LH, Yang YJ (1995) Schwann cell properties: 3. C-fos expression, bFGF 32. Van Rooijen N, Sanders A (1994) Liposome mediated depletion of macrophages: production, phagocytosis and proliferation during Wallerian degeneration. Mechanism of action, preparation of liposomes and applications. J Immunol Methods J Neuropathol Exp Neurol 54:487–496. 174:83–93. 13. Perry VH, Tsao JW, Fearn S, Brown MC (1995) Radiation-induced reductions in mac- 33. Romanelli E, et al. (2016) Myelinosome formation represents an early stage of oligo- rophage recruitment have only slight effects on myelin degeneration in sectioned dendrocyte damage in multiple sclerosis and its animal model. Nat Commun 7:13275. – peripheral nerves of mice. Eur J Neurosci 7:271 280. 34. Brosius Lutz A, Barres BA (2014) Contrasting the glial response to axon injury in the 14. Arthur-Farraj PJ, et al. (2012) c-Jun reprograms Schwann cells of injured nerves to central and peripheral nervous systems. Dev Cell 28:7–17. – generate a repair cell essential for regeneration. Neuron 75:633 647. 35. Cahoy JD, et al. (2008) A transcriptome database for astrocytes, neurons, and oligo- 15. Napoli I, et al. (2012) A central role for the ERK-signaling pathway in controlling dendrocytes: A new resource for understanding brain development and function. Schwann cell plasticity and peripheral nerve regeneration in vivo. Neuron 73:729–742. J Neurosci 28:264–278. 16. Peleshok JC, Ribeiro-da-Silva A (2012) Neurotrophic factor changes in the rat thick 36. Safaiyan S, et al. (2016) Age-related myelin degradation burdens the clearance skin following chronic constriction injury of the sciatic nerve. Mol Pain 8:1. function of microglia during aging. Nat Neurosci 19:995–998. 17. Mehlem A, Hagberg CE, Muhl L, Eriksson U, Falkevall A (2013) Imaging of neutral 37. Liddelow SA, et al. (2017) Neurotoxic reactive astrocytes are induced by activated lipids by oil red O for analyzing the metabolic status in health and disease. Nat Protoc – 8:1149–1154. microglia. Nature 541:481 487. 18. Klionsky DJ, et al. (2012) Guidelines for the use and interpretation of assays for 38. Zamanian JL, et al. (2012) Genomic analysis of reactive astrogliosis. J Neurosci 32: – monitoring autophagy. Autophagy 8:445–544. 6391 6410. 19. Mizushima N, Kuma A (2008) Autophagosomes in GFP-LC3 transgenic mice. Methods 39. Lutz AB (2014) Purification of Schwann cells from the neonatal and injured adult Mol Biol 445:119–124. mouse peripheral nerve. Cold Spring Harb Protoc 2014:1312–1319. 20. Jang SY, et al. (2016) Autophagic myelin destruction by Schwann cells during 40. Ma CH, et al. (2011) Accelerating axonal growth promotes motor recovery after pe- Wallerian degeneration and segmental demyelination. Glia 64:730–742. ripheral nerve injury in mice. J Clin Invest 121:4332–4347. 21. Komatsu M, et al. (2005) Impairment of starvation-induced and constitutive auto- 41. Möbius W, et al. (2010) Electron microscopy of the mouse central nervous system. phagy in Atg7-deficient mice. J Cell Biol 169:425–434. Methods Cell Biol 96:475–512.

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