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doi: 10.1242/10.1242/jcs.00046

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Retinoic acid receptor ß2 and neurite outgrowth in the adult mouse spinal cord in vitro

Jonathan Corcoran1,*, Po-Lin So1,*, Robert D. Barber2, Karen J. Vincent2, Nicholas D. Mazarakis2, Kyriacos A. Mitrophanous2, Susan M. Kingsman2 and Malcolm Maden1,{ddagger}

1 MRC Centre for Developmental Neurobiology, New Hunt's House, King's College London, Guy's Campus, London SE1 1UL, UK
2 Oxford BioMedica (UK) Limited, Medawar Centre, Robert Robinson Avenue, The Oxford Science Park, Oxford OX4 4GA, UK



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  		Fig. 1. Induction of neurite outgrowth by retinoic acid. Comparison of the effect of retinoic acid in neurite outgrowth in cultured embryonic day 13.5 mouse spinal cord (A-C) and 10-month-old adult mouse spinal cord (D-F). Pieces of spinal cord were cultured in cellagen in the presence of 10% delipidated serum with or without RA for a period of 5 days. The medium was changed every 2 days. In A and D there is no RA. The embryonic cord extends neurites in the absence of RA, whereas the adult cord does not. In B and E, 1x10-6M RA was added. Many more neurites are extended from the embryonic cord after RA addition, whereas the adult cord is still unresponsive. C and F show RT-PCR analysis of cultured cords to analyze the expression of RARß2 after after 5 days. The presence of GAPDH was used to indicate equal amounts of cDNA in the samples. (C) Embryonic day 13.5 cord. Lane 1, no RA; lane 2, 1x10-8 M RA; lane 3, 1x10-7 M RA, lane 4, 1x10-6 M RA. RARß2 is upregulated by RA. (F) Adult cord. Lane 1, no RA; lane 2, 1x10-8 M RA; lane 3, 1x10-7 M RA, lane 4, 1x10-6 M RA. RARß2 fails to be upregulated at any concentration of RA. Arrows on the right of F show the position of GAPDH and RARß2. (G) A western blot of proteins from embryonic mouse spinal cord (lane 1) and adult mouse spinal cord (lane 2) incubated with a RARß2 antibody. This part of the blot, at a molecular weight of approximately 45 kDa, confirms the presence of RARß2 protein in embryonic but not adult cord.

 


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  		Fig. 3. Viral transduction of spinal cord. (A) Transduction of adult mouse spinal cord with pONY8.0cRARß2. This culture was analyzed 8 days post-transduction with a FITC-labelled anti-Flag antibody (green). The area of green fluorescence shows a high level of transduction within the cultured cord. (B) The effect of transducing adult mouse spinal cord with pONY8.0cRARß2. Neurites, stained with an antibody against NF-200 and visualized with diaminobenzidine can be seen growing out from the explant (ex) after 8 days. (C) Light cycler PCR analysis for RARß2 expression in three individual RARß2 transduced explants (lanes 1-3) and three individual lacZ control transduced explants (lanes 4-6) cultured for 8 days. The levels of RARß2 expression relative to GAPDH are recorded in the blue columns. Explants 1-3 show between a 100-fold and a 300-fold induction of RARß2, whereas explants 4-6 show no expression. (D) Double-labelled adult mouse cord explant following transduction with pONY8.0cRARß2. Neurites were stained with NF-200 and visualized with a Texas Red-labelled secondary antibody and can be seen emerging from the lower part of the explant. Transduced cells were visualized with a FITC-labelled anti-Flag antibody (green). Double-labelled cells (yellow) are transduced cells that are also NF-200 positive. (E) Close-up of the explant in D showing green RARß2-transduced cells, red neurofilament-positive cells and yellow double-stained cells. (F) Adult mouse cord transduced with the control virus (pONY8Z) containing the lacZ marker gene. After X-gal staining, a good percentage of transduced cells (mostly neurons as judged by their size) is obtained. (G) The effect of transducing adult mouse cord with a herpes virus containing the RARß4 gene (pHSVRARß4). No neurites are induced. (H) The effect of transducing adult cord with a herpes virus containing the RARß2 gene (pHSVRARß2). In contrast to G, neurites are induced in this case, as visualized with NF-200 and diaminobenzidine. (I) RT-PCR analysis of adult mouse cords transduced with either the RARß2 gene (pHSVRARß2) or the RARß4 gene (pHSVRARß4 — lanes 5-7) and analyzed after 4 days. Lanes 1-3, RARß2 analysis: lane 1, no virus; lane 2, pHSVRARß2 transduction showing the presence of RARß2; lane 3, pHSVRARß4 transduction showing no RARß2 expression. Lanes 5-7, RARß4 analysis: lane 5, no virus; lane 6, pHSVRARß2 transduction showing the absence of RARß2; lane 7, pHSVRARß4 transduction showing the presence of RARß4 expression. Arrows to the right mark the position of RARß2, RARß4 and GAPDH.

 


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  		Fig. 2. Neuronal transduction using EIAV. (A,B) Brightfield (A) and fluorescent (B) image of dissociated dorsal root ganglia transduced with pONY8.0G. At a multiplicity of infection (MOI) of 10, 45% of cells in this culture were transduced as shown by GFP fluorescence in B. (C-E) Confocal microscopy images of the same two cells in primary cultures of striatal neurons transduced with the EIAV construct, pONY8RARß2.Flag. In C a RARß antibody and in D a Flag antibody were used to visualize transduced cells with Texas-Red- and FITC-coupled secondary antibodies, respectively. In E, the images are merged to show the colocalization (yellow) of the RARß and Flag antibody staining.

 


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  		Fig. 4. Presence of retinoids in adult mouse spinal cord. A chromatogram showing two peaks. Peak one has a UV absorbance maximum at 342 nm, and on the basis of its spectrum is identified as all-trans-RA. Peak two has a UV absorbance maximum at 324nm and is identified as all-trans-retinol and/or 13-cis-retinol.

 

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