A LewisX glycoprotein screen identifies the low density lipoprotein receptor-related protein 1 (LRP1) as a modulator of oligodendrogenesis in mice

Abstract

In the developing and adult CNS multipotent neural stem cells reside in distinct niches. Specific carbohydrates and glycoproteins are expressed in these niche microenvironments which are important regulators of stem cell maintenance and differentiation fate. LewisX (LeX), also known as stage-specific embryonic antigen-1 or CD15, is a defined carbohydrate moiety expressed in niche microenvironments of the developing and adult CNS. LeX-glycans are involved in stem cell proliferation, migration, and stemness. A few LeX carrier proteins are known, but a systematic analysis of the targets of LeX glycosylation in vivo has not been performed so far. Using LeX glycosylation as a biomarker we aimed to discover new glycoproteins with a potential functional relevance for CNS development. By immunoaffinity chromatography we enriched LeX glycoproteins from embryonic and postnatal mouse brains and used one-dimensional nLC-ESI-MS/MS for their identification. We could validate phosphacan, tenascin-C, and L1-CAM as major LeX carrier proteins present in vivo. Furthermore, we identified LRP1, a member of the LDL receptor family, as a new LeX carrier protein expressed by mouse neural stem cells. Surprisingly, little is known about LRP1 function for neural stem cells. Thus, we generated Lrp1 knock-out neural stem cells by Cre-mediated recombination and investigated their properties. Here, we provide first evidence that LRP1 is necessary for the differentiation of neural stem cells toward oligodendrocytes. However, this function is independent of LeX glycosylation.

Keywords: Carbohydrate; Development; Differentiation; Extracellular Matrix Proteins; Glycoprotein; Lipoprotein-like Receptor (LRP); Neural Stem Cell.

FIGURE 1.

LeX carrier proteins change during mouse CNS development. A, Western blot with anti-LeX mAb 5750^LeX of brain lysates at the indicated developmental stages. Note that the detected LeX-positive proteins shift from embryonic to postnatal stages. B, Western blot against LeX of E14 cortex (Ctx) and ganglionic eminence (GE). C, at E13 most LeX-positive proteins are membrane-associated, whereas at P12 LeX proteins accumulate in the membrane-free (supernatant) fraction after differential centrifugation. D–G, LeX immunostainings of E12.5 (E) or P1 (G) coronal forebrain sections depicted in D and F. L/MGE, lateral/medial GE; Di, diencephalon; LV, lateral ventricle; S, septum, α-tub., α-tubulin. Scale bars, 200 μm

FIGURE 2.

Mass spectrometric identification of LRP1 as a novel LeX glycoprotein. A, experimental setup for the purification of LeX-positive proteins from mouse brains by immunoaffinity chromatography and subsequent identification via one-dimensional nLC-ESI-MS/MS. B, tabular summary of known LeX carrier proteins and LRP1 identified in embryonic and postnatal tissue preparations, listing the peptide counts in each of the seven independently analyzed protein samples. C and D, LeX-positive proteins immunopurified from embryonic or postnatal brains using either anti-LeX mAbs 487^LeX or 5750^LeX were separated by SDS-PAGE and analyzed by Western blotting or silver staining. Prominent protein bands were excised from the gel and analyzed via one-dimensional nLC-ESI-MS/MS. Note that some LeX carrier proteins such as Ptprz1 expose glycosaminoglycan chains and do not focus into distinct bands in SDS-PAGE. However, these constituents appear as high molecular smear on immunoblots.

FIGURE 3.

LRP1 is expressed by neural stem cells in the embryonic cortex. A–C, E12.5, coronal plane, and cortical sections immunostained against LRP1 (green) and LeX (red) (A and B), or LRP1 (red) and Nestin (green) (C). Note that LRP1 co-localizes with stem cell markers on a subpopulation of radial cells (arrow). D, coronal forebrain section at E14.5 stained for LRP1 (green) and Ctip2 (red) labeling layer VI/V neurons. E and F, acutely dissociated E14.5 cortical cells plated on laminin for 2 h and stained against the indicated markers. Note that some cells with pronounced LRP1 expression do not express stem cell markers (arrowhead). G and H, LRP1 staining (green) at P1 in cortex (G) and lateral ventricle (H). For orientation, sections were co-stained against Ctip2 (G) or LeX (H). Note the up-regulation of LRP1 protein in the marginal zone (G) and adjacent to the lateral ventricle (H). I, magnification of the boxed area in H depicting LRP1- and LeX-double positive cells at the lateral ventricle. In E, F, and H, nuclei are stained with Hoechst (blue). ChPl, choroid plexus; ctx, cortex; M/LGE, medial/lateral ganglionic eminence; HP, hippocampal anlage; Di, diencephalon; SVZ, subventricular zone; LV, lateral ventricle. Scale bars, 100 μm (A–D, G, and H); 25 μm (E, F, and I).

FIGURE 4.

NSPCs express LeX glycoforms of LRP1. A, LRP1 domain structure. Note that LRP1 α- and β-chain are noncovalently linked and run as separate subunits in SDS-PAGE. B, immunoprecipitation (IP) of LeX from NSPCs cultivated as neurospheres. The Western blot against LeX reveals multiple LeX-positive proteins in NSPC input protein lysates. LRP1 precipitates with LeX-positive proteins shown by the detection of LRP1 β-chain. Arrowheads label unspecific antibody signals. C, LRP1 immunoprecipitation from NSPCs yielding a single LeX-positive protein with a size correlating to the expected size of the LRP1 α-chain (asterisk). LRP1 precipitation was confirmed by detection of LRP1 β-chain. D and E, RAP-mediated affinity purification of LRP1 from NSPC (D) or P1 brain (E) lysates. The RAP ligand is positive for LeX and HNK-1. F, Western blots after antibody-mediated immunoprecipitation of LRP1 and subsequent N-glycanase F (N-Glyk. F) treatment. Note that LeX and HNK-1 immunoreactivity on LRP1 α-chain are lost after enzymatic removal of N-linked glycans.

FIGURE 5.

Lrp1 knock-out NSPCs proliferate and differentiate in vitro. A, schematic diagram outlining the generation of Lrp1 knock-out NSPCs. NSPCs obtained from Lrp1^flox/flox mice or Lrp1^wt/wt littermates were treated with cell-permeant Cre recombinase, expanded as neurospheres and differentiated. B, scheme of the Lrp1 gene locus illustrating the location of the loxP sites and the primers used to assess recombination. C, exemplary PCR verifying successful recombination in Cre-treated cells. D, phase contrast image of neurospheres derived from Cre-treated Lrp1^flox/flox or Lrp1^wt/wt cells. E, Western blot demonstrating the reduction of LRP1 protein in NSPCs after recombination. F, LRP1 expression permanently eliminated in Cre-treated Lrp1^flox/flox NSPCs even when passaged over a time period of 6 weeks. G–J, immunostainings against cell type-specific biomarkers after differentiation of Lrp1 knock-out. Nestin (neural stem precursor cells (NSPCs)); GFAP, glial fibrillary acidic protein (astrocytes); O4 (oligodendrocytes); βIII-tubulin, (young neurons). Scale bar, 50 μm.

FIGURE 6.

Lrp1 knock-out inhibits oligodendroglial differentiation. A–D, immunolabeling of oligodendrocytes by O4 (A and C) in Lrp1 wild-type (Lrp1^wt/wt), or knock-out (Lrp1^flox/flox) NSPC cultures, differentiated for 7 days. The corresponding Hoechst stained nuclei are shown in B and D. E, quantification of O4-positive cells in wild-type (wt/wt) or knock-out (flox/flox) cultures. F, differentiation of wild-type NSPCs in the presence of LeX-epitope blocking mAb 487^LeX. The anti-Lex treatment does not influence the percentage of O4-positive cells compared with PBS-treated control. G, differentiation of wild-type NSPCs in the presence of RAP reducing the amount of O4-positive cells compared with GST-treated control. Scale bar, 75 μm; ns, not significant. Data are expressed as mean ± S.D. (error bars), n = 4; **, p ≤ 0.01.