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. 2016 Aug;64(8):1363-80.
doi: 10.1002/glia.23009. Epub 2016 Jun 3.

Low-density lipoprotein receptor-related protein 1 is a novel modulator of radial glia stem cell proliferation, survival, and differentiation

Dina Safina  1   2 , Frederik Schlitt  1 , Ramona Romeo  1 , Thorsten Pflanzner  3 , Claus U Pietrzik  3 , Vasanthy Narayanaswami  4 , Frank Edenhofer  5 , Andreas Faissner  1
Affiliations

Low-density lipoprotein receptor-related protein 1 is a novel modulator of radial glia stem cell proliferation, survival, and differentiation

Dina Safina et al. Glia. 2016 Aug.

Abstract

The LDL family of receptors and its member low-density lipoprotein receptor-related protein 1 (LRP1) have classically been associated with a modulation of lipoprotein metabolism. Current studies, however, indicate diverse functions for this receptor in various aspects of cellular activities, including cell proliferation, migration, differentiation, and survival. LRP1 is essential for normal neuronal function in the adult CNS, whereas the role of LRP1 in development remained unclear. Previously, we have observed an upregulation of LewisX (LeX) glycosylated LRP1 in the stem cells of the developing cortex and demonstrated its importance for oligodendrocyte differentiation. In the current study, we show that LeX-glycosylated LRP1 is also expressed in the stem cell compartment of the developing spinal cord and has broader functions in the developing CNS. We have investigated the basic properties of LRP1 conditional knockout on the neural stem/progenitor cells (NSPCs) from the cortex and the spinal cord, created by means of Cre-loxp-mediated recombination in vitro. The functional status of LRP1-deficient cells has been studied using proliferation, differentiation, and apoptosis assays. LRP1 deficient NSPCs from both CNS regions demonstrated altered differentiation profiles. Their differentiation capacity toward oligodendrocyte progenitor cells (OPCs), mature oligodendrocytes and neurons was reduced. In contrast, astrocyte differentiation was promoted. Moreover, LRP1 deletion had a negative effect on NSPCs proliferation and survival. Our observations suggest that LRP1 facilitates NSPCs differentiation via interaction with apolipoprotein E (ApoE). Upon ApoE4 stimulation wild type NSPCs generated more oligodendrocytes, but LRP1 knockout cells showed no response. The effect of ApoE seems to be independent of cholesterol uptake, but is rather mediated by downstream MAPK and Akt activation. GLIA 2016 GLIA 2016;64:1363-1380.

Keywords: LRP1; LRP1-dependent signaling; LewisX carbohydrate; membrane permeant Cre-recombinase; neural stem and progenitor cells; neural stem cell differentiation.

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Figures

Fig. 1
Fig. 1. LRP1 is LewisX glycosylated in the developing spinal cord
A. An overview showing frontal spinal cord sections of E12.5 C57Bl6 mouse embryos immunostained against the glycan LewisX epitope mAb5750LeX and mAb487LeX (red) co-localizing with LRP1 (green) and Nestin (blue) in the radial glia cells of the central canal, scale bar 100 μm. B. High magnification images of the central canal region, immunostained for the same markers as in A, confirming co-localization, scale bar 50 μm. C. Western blot detecting LRP1α-chain, 5750LeX and 487LeX epitopes in the E14.5 spinal cord protein lysate, immunoprecipitated with mAb B411E2, illustrating the LeX glycosylation of mature LRP1 in the developing spinal cord. IP = immunoprecipitation, AB = antibody.
Fig. 2
Fig. 2. LRP1 deletion interferes with NSPC proliferation and survival
A. Representative photomicrographs of LRP1+/+ and LRP1−/− NSPCs immunostained against BrdU (N = 7, cx; N = 10, sc) and apoptosis marker active Caspase3 (N = 9, cx; N = 11, sc). Scale bar 50 μm. B. Quantification results indicate 1.5 fold reduction in proliferation and 2 fold increase in the apoptosis rate of LRP1 NSPCs derived from cortex and spinal cord. Data are expressed as mean ± SE (* indicates P < 0.05, ** — P < 0.01). Cx = cortex, sc = spinal cord.
Fig. 3
Fig. 3. LRP1 deletion impairs lineage progression of oligodendrocytes
Representative photomicrographs of LRP1+/+ and LRP1 differentiated NSPCs derived from cortex and spinal cord immunostained against: A. PDGFRα — a marker of OPCs (N = 5, cx; N = 13, sc). LRP1−/− cells generate 2 times less OPCs. B. Sulfatide O4 — a marker of differentiated immature oligodendrocytes (N = 5, cx; N = 10, sc). LRP1−/− cells generate 2 times less immature oligodendrocytes. C. MBP — a marker of mature oligodendrocytes (N = 4, cx). LRP1−/− cells generate 3 times less mature oligodendrocytes. Scale bar 50 μm. Data are expressed as mean ± SE (* indicates P < 0.05, ** — P < 0.01). Cx = cortex, sc = spinal cord
Fig. 4
Fig. 4. LRP1 knockout NSPCs generated less neurons, but more astrocytes
Representative photomicrographs of LRP1+/+ and LRP1−/−differentiated NSPCs derived from cortex and spinal cord immunostained against: A. βIII-tubulin — a marker of young neurons (N = 10, cx, N = 10, sc). LRP1−/− NSPCs derived from both tissues generate nearly twice less neurons. B. GFAP — an astrocytic marker (N = 7, cx; N = 14, sc). LRP1 NSPCs derived from both tissues generate around 1.5 times more astrocytes. Scale bar 50 μm. Data are expressed as mean ± SE (* indicates P < 0.05, ** — P < 0.01). Cx = cortex, sc = spinal cord.
Fig. 5
Fig. 5. Western blot confirms the modulation of NSPC differentiation by LRP1 deletion
A. Representative Western blot analysis of protein lysates from differentiated NSPCs detecting LRP1, two house keeping genes: α-tubulin and actin and cell specific markers: PDGFRα, GFAP and βIII-tubulin. Quantification of Western blot results reveals a reduction of OPCs marker PDGFRα (B), neuronal marker βIII-tubulin (C), and an increase of GFAP expression in LRP1−/− cells. N=5, data are expressed as mean ± SE (* indicates P < 0.05, ** — P < 0.01).
Fig. 6
Fig. 6. LRP1 deletion modifies neuronal polarity
A. Representative photomicrographs of LRP1+/+ and LRP1−/− differentiated NSPCs derived from cortex immunostained against βIII-tubulin. The longer neurites (presumably axons) and the shorter processes (presumably dendrites) are indicated with green and red false color traces, respectively. B. The comparison of total neurite, dendrite and axon length between LRP1+/+ and LRP1−/− neurons. LRP1 knockout neurons exhibit significantly shorter dendrites and longer axons compared to wild type neurons, indicating alterations in neuronal polarity upon LRP1 deletion.
Fig. 7
Fig. 7. ApoE promotes oligodendroglial differentiation via LRP1
The results of the differentiation assay: A. illustrating that lipid-free ApoE4 addition to the differentiation medium increases the amount of O4-positive oligodendrocytes generated from LRP1+/+ (N = 4). This suggests that LRP1-dependent ApoE uptake promotes the differentiation of NSPCs towards oligodendrocytes. B. Oligodendroglial differentiation of LRP1−/− NSPCs is not altered upon ApoE4 treatment. Data are expressed as mean ± SE (* indicates P < 0.05, ** — P < 0.01).
Fig. 8
Fig. 8. Cholesterol is important for oligodendroglial differentiation but cholesterol levels are not affected by LRP1 deletion
A. Representative live confocal fluorescent images of NSPCs after TopFluor cholesterol accumulation and subsequent cholesterol extraction with MβCD demonstrate the effectiveness of the MβCD-treatment. Scale bar 20 μm. B. The results of the differentiation assay show that MβCD-treatment has a negative effect on oligodendroglia, decreasing the percentage of O4-positive oligodendrocytes generated from wild type NSPCs. This suggests that cholesterol is involved in oligodendrogenesis (N = 7). C. Representative live confocal fluorescent images of adherent proliferating NSPCs after rHDL/TopFluor/apoE4 cholesterol accumulation and the quantification of fluorescence intensity. D. Quantification of rHDL/TopFluor/apoE4 fluorescent intensity shows no difference in cholesterol uptake between LRP1−/− and LRP1+/+ cells (N = 4). Scale bar 50 μm. Total cholesterol concentration measured with the AmplexRed cholesterol kit does not differ between LRP1 and LRP1+/+ NSPCs (N = 16). Data are expressed as mean ± SE, (* indicates P < 0.05, ** — P < 0.01).
Fig. 9
Fig. 9. Wild type but not NPXY mutant mini LRP1 is able to rescue oligodendroglial differentiation in LRP1 cells
A. Schematic representation of full-length receptor LRP1, wild type and mutated mini-receptors. I-IV = the ligand binding domains. B. A representative fluorescent image of neurospheres electroporated with GFP, illustrating high transfection efficiency. The result of the differentiation assays shows that LRP1 knockout NSPCs transfected with wild type mini LRP1 generated significantly more O4-positive oligodendrocytes compared to NSPCs transfected with mini-LRP1 including the mutated intracellular domain, or GFP-transfected cells (N = 4). Data are expressed as mean ± SE, (* indicates P < 0.05, ** — P < 0.01).
Fig. 10
Fig. 10. LRP1 promotes ERK and Akt phosphorylation upon ApoE stimulation
Representative Western blot analysis and quantification, showing that ApoE can enhance ERK1/2 (A) and Akt phosphorylation (B) in LRP1+/+ but not LRP−/− cells (N = 6, Akt; N = 7, ERK). The band intensity of p-Akt and p-ERK is normalized to total Akt and ERK respectively. Data are expressed as mean ± SE (* indicates P < 0.05, ** — P < 0.01). p-ERK = phosphorylated ERK, p-Akt = phosphorylated Akt, t-ERK = total ERK, t-Akt = total Akt.
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References

    1. Alvarez-Buylla A, García-Verdugo JM. Neurogenesis in adult subventricular zone. The Journal of Neuroscience. 2002;22:629–634. - PMC - PubMed
    1. Baron W, Bijlard M, Nomden A, de Jonge JC, Teunissen CE, Hoekstra D. Sulfatide-mediated control of extracellular matrix-dependent oligodendrocyte maturation. Glia. 2014;62:927–42. - PubMed
    1. Bertram B, Wiese S, von Holst A. High-efficiency transfection and survival rates of embryonic and adult mouse neural stem cells achieved by electroporation. Journal of Neuroscience Methods. 2012;209:420–427. - PubMed
    1. Bieberich E. It’s a lipid's world: Bioactive lipid metabolism and signaling in neural stem cell differentiation. Neurochemical Research. 2012;37:1208–1229. - PMC - PubMed
    1. Blaschuk KL, Frost EE, ffrench-Constant C. The regulation of proliferation and differentiation in oligodendrocyte progenitor cells by alphaV integrins. Development. 2000;127:1961–9. - PubMed

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