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First published online 19 March 2009
doi: 10.1242/jcs.040717

Journal of Cell Science 122, 1155-1162 (2009)
Published by The Company of Biologists 2009
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Low-density lipoprotein receptor-related protein 1 is an essential receptor for myelin phagocytosis

Alban Gaultier1,*, Xiaohua Wu2,*, Natacha Le Moan3,4, Shinako Takimoto1, Gatambwa Mukandala1, Katerina Akassoglou3,4, W. Marie Campana5 and Steven L. Gonias1,{ddagger}

1 Department of Pathology, University of California, San Diego, La Jolla, CA 92093, USA
2 The McColl-Lockwood Laboratory for Muscular Dystrophy Research, Cannon Research Center, Carolinas Medical Center, Charlotte, NC 28203, USA
3 Gladstone Institute of Neurological Diseases, University of California, San Francisco, San Francisco, CA 94158, USA
4 Department of Neurology, University of California, San Francisco, San Francisco, CA 94143, USA
5 Department of Anesthesiology, University of California, San Diego, La Jolla, CA 92093, USA


Figure 1
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  		Fig. 1. LRP1 is a receptor for myelin vesicles. (A) MVs were analyzed by SDS-PAGE with Coomassie staining and by immunoblot analysis using MBP-specific antibody. (B) PEA-10 and MEF-2 cells were incubated with FITC-labeled MVs for 30 minutes in the presence of GST or GST-RAP. After washing and protease treatment, cells were subjected to flow cytometry analysis. (C) PEA-10 and MEF-2 cells were incubated with Rhodamine-labeled MVs for 30 minutes. After washing, the cells were incubated with Lysotracker for 30 minutes and analyzed by fluorescence microscopy. Scale bar: 50 µm.

 

Figure 2
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  		Fig. 2. CNS glia express LRP1. (A) RNA was isolated from oligodendrocytes (Oligo), astrocytes (Astro) and microglia (Micro). LRP1 mRNA expression was determined by qPCR (data are means ± s.d.). (B) Protein extracts from oligodendrocytes, astrocytes and microglia were analyzed by immunoblotting with LRP1 β-chain-specific antibody 11H4 and with antibody specific for ERK/MAP kinase as a control for loading. The same samples were also analyzed by RAP-ligand blotting to detect LRP1 {alpha}-chain and possibly other LRP family members. (C) MVs were analyzed by immunoblot analysis using specific antibodies that detect MBP or LRP1 β-chain and by RAP ligand blotting. Oligodendrocyte extracts were assessed in the same experiments.

 

Figure 3
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  		Fig. 3. MV phagocytosis by CNS glia is inhibited by RAP. Oligodendrocytes (A) and astrocytes (B) were incubated with FITC-labeled MVs for 30 minutes in the presence of GST or GST-RAP. After washing and protease treatment to dissociate surface-associated MVs, the cells were subjected to flow cytometry analysis. (C) Microglia were incubated with Rhodamine-labeled MVs for 30 minutes in the presence of GST or GST-RAP. After washing, the cells were analyzed by fluorescence microscopy and by phase contrast microscopy. Scale bar: 50 µm.

 

Figure 4
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  		Fig. 4. LRP1 gene-silencing blocks MV uptake by oligodendrocytes. Oligodendrocytes were transiently transfected with NTC or LRP1-specific siRNA. (A) LRP1 expression at the protein level was assessed 36 hours after silencing by immunoblot analysis with LRP1-specific antibody, 11H4. Blots were also probed for tubulin as a control for load. (B) Oligodendrocytes were incubated with FITC-labeled MVs for 30 minutes. After washing and protease treatment, internalized MVs were detected by flow cytometry.

 

Figure 5
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  		Fig. 5. MBP binds to LRP1 and mediates MV uptake. (A) rMBP was expressed as a His-tagged fusion protein in bacteria and subjected to SDS-PAGE with Coomassie staining and immunoblot analysis. (B) Purified rat LRP1 and fibronectin were subjected to SDS-PAGE and electrotransferred to PVDF membranes. The membranes were probed with 125I-labeled rMBP in the presence or absence of MBP-specific antibody. (C) BSA, rMBP and GST-RAP were adsorbed onto plastic wells and incubated with shed LRP1 in the presence of GST-RAP or GST. LRP1 binding to the immobilized phase was detected in an ELISA format, using LRP1 {alpha}-chain-specific antibody 8G1. (D) FITC-labeled MVs were incubated with oligodendrocytes in the presence of MBP-specific IgG or non-immune IgG. MV internalization was determined by flow cytometry.

 

Figure 6
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  		Fig. 6. LRP1 expression in EAE. (A) Protein extracts of the cerebellum and spinal cord of mice that were treated to induce EAE, and from control mice, were subjected to immunoblot analysis to detect LRP1, using the LRP1 β-chain-specific antibody, 11H4. The same blots were also probed to detect tubulin as a control for loading. (B) Sagittal brain section from mice with EAE, and control mice, were stained to detect the microglia/macrophage marker, IsoB4 (green) and LRP1 (red). Equivalent regions of the brains of control and EAE mice are shown. The insets show a macrophage/microglial cell infiltrate at higher magnification. Scale bar, 200 µm. (C) Sagittal brain section were immunostained to detect astrocytes (GFAP, red) and LRP1 (green). Images of the hippocampus are shown. The insets show astrocytes that are immmunopositive for both GFAP and LRP1. Scale bar, 200 µm. (D) Sequential sagittal brain sections were immunostained for oligodendrocytes (CNPase) and LRP1. Scale bar: 50 µm.

 

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