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Science 339 (6117): 324-328

Copyright © 2013 by the American Association for the Advancement of Science

GDE2 Promotes Neurogenesis by Glycosylphosphatidylinositol-Anchor Cleavage of RECK

Sungjin Park1,*, Changhee Lee1,*, Priyanka Sabharwal1, Mei Zhang1, Caren L. Freel Meyers2, and Shanthini Sockanathan1,{dagger}

1 Solomon Snyder Department of Neuroscience, School of Medicine, Johns Hopkins University, PCTB1004, 725 N Wolfe Street, Baltimore, MD 21205, USA.
2 Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, WBSB 301A, 725 N Wolfe Street, Baltimore, MD 21205, USA.


Figure 1
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Fig. 1. Stimulation of Dll1 shedding by GDE2. (A to H) Coronal sections of embryonic day 13 (E13.5) mouse spinal cords. Arrows mark V2b interneurons (red). (I) Graph quantifying interneuron numbers in WT and Gde2–/– mutants; mean ± SEM, n = 4 embryos, two-tailed t test: V0, *P = 0.0016; V1, P = 0.4778; V2a, *P = 0.0028; V2b, *P = 0.0088. (J) Western blots of extracts of chick spinal cords electroporated with Dll1-Flag plasmid; in the top blot, the open arrow indicates 30-kD Dll1 C-terminal fragment, and the solid arrow indicates C-terminal 42-kD Dll1 product (Dll1-42). In the top middle blot, the solid arrow (GDE2) indicates endogenous glycosylated GDE2, and the bottom bands are hypoglycosylated GDE2. (K) Western blot of Jag1 processing (FL, full length; CTF, C-terminal fragment) and quantification of Jag1 CTF/FL ratios from E12.5 embryonic spinal cord extracts. (L to N) Close-up of electroporated chick spinal cords (right) shows increased Isl2+ MNs (red) when Dll1 is coelectroporated with GDE2. Arrows indicate midline. Scale bar, 20 μm.

 

Figure 2
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Fig. 2. Effects of RECK ablation. (A to F) Notch target gene mRNAs are reduced in HH stages 19/20 chick spinal cords electroporated with RECK shRNAs but not control shRNAs (CshRNAs). (G to L) Olig2 expression (blue) demarcates VZ of chick spinal cords electroporated (right) with control and RECK shRNAs, showing Isl2+ MNs (red) that express neuronal Tuj1 (green) when RECK is knocked down. Arrows indicate midline; doubleheaded arrows indicate VZ. Scale bar, 20 μm. (M) Western blots of chick spinal cords electroporated with Dll1-Flag plasmid and RECK shRNAs show RECK knockdown stimulates Dll1-42 production (arrow), but Jag1 expression and processing is unchanged. Shown is a graph quantifying Dll1-42 cleavage from Western blots; mean ± SEM. Two-tailed t test, n = 4 embryos; sh1RECK *P = 0.0066; sh2RECK *P = 0.0175.

 

Figure 3
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Fig. 3. GDE2 cleaves RECK within the GPI-anchor. (A) Schematic of two-step GDPD catalysis. (B) Graph quantifying in vitro GDPD assay in transfected HEK293T cells using glycerophosphoserine (GPSerine) and synthetic cyclic glycerol[1,2] phosphate intermediate. v, empty vector. (C to E) Western blots of transfected HEK293T cell lysates (lys) and medium (med). (C) RECK is detected in the medium when catalytically active GDE2 is present. (D) After sequential Triton X-114 extraction cleaved, RECK is observed in the Detergent (DT)–free hydrophilic phase, whereas Dll1, which is not cleaved by GDE2, is retained in the DT-rich hydrophobic phase of the lysate. (E) RECK ECD is generated by GDE2 or PI-PLC activities. (F) Western blot of lysates (lys) and medium (med) of HEK293T cells transfected with RECK and C-terminal Flag-tagged GDE2 or GPI-PLD. Surface RECK is labeled by biotin. GDE2 but not GPI-PLD releases surface-biotinylated RECK into the medium (arrow). Both GDE2 and GPI-PLD are visualized by antibodies to Flag, but only GDE2 is labeled by biotin, indicating that GDE2 is localized to the cell surface. PI-PLC was added to intact cells and serves as a positive control. (G) Schematic of GPI-anchor (top) and graph quantifying amount of radiolabel incorporated into RECK or sRECK when GDE2 or PI-PLC is present (bottom). Mean ± SEM; n = 4 to 12 samples.

 

Figure 4
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Fig. 4. GDE2 inactivates RECK through GPI-anchor cleavage. (A and B) Graphs quantifying ratio of ectopic Isl2+ VZ MNs normalized to the number of transfected GDE2 cells. Mean ± SEM, two-tailed t test. (A) Suboptimal levels of plasmids expressing RECKs/opt or RECK-CD2s/opt were coelectroporated with GDE2. RECK-CD2 was more effective than was RECK in suppressing GDE2-dependent MN generation. *P = 0.0306; n = 5 embryos. (B) Plasmids expressing RECK or sRECK were coelectroporated with GDE2; RECK effectively suppressed GDE2 function, but sRECK did not (*P = 5.59 x 10–5; n = 8 to 10 embryos) as compared with GDE2. (C) Western blot of extracts of chick spinal cords electroporated with Dll1-Flag and RECK shRNA targeting 3' untranslated region to detect full-length (FL) and processed Dll1-42. The phenotype is rescued by exogenous plasmids expressing WT RECK open reading frame but not sRECK. Densitometric quantification of Dll1-42, mean ± SEM, n = 4 embryos. Two-tailed t test, *P = 0.013 compared with empty.

 


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