Figure 3: Potential mediators of angiogenesis defects in Glut1 DS and a functional evaluation of Glut1 constructs for gene replacement.

(a) A quantification of Vegfr2 RNA levels demonstrates reduced transcripts specifically in the capillaries of the Glut1 DS brain. (b) Western blot analysis and (c) a quantification of Vegfr2 protein in the capillaries (Cap) and the capillary-depleted brain (CDB) fraction also depict reduced protein in mutant brain vessels. Also shown in the blot are corresponding Glut1 levels which are particularly high in the capillaries (55 kDa isoform) compared with the CDB fraction (45 kDa isoform). Results of high as well as low exposure times are depicted. (d) Basal glycolytic flux and maximal respiration are both significantly compromised in cells from Glut1 DS model mice. Panels a–d: *, **, P<0.05, P<0.01, t-test, N≥3 samples, 2 independent preparations. (e) Western blot depicting an increase in Glut1 protein following transfection of Glut1 DS patient fibroblasts with either a murine (lane 2) or human (lane 3) Glut1 cDNA construct. The ubiquitously expressed SMN protein was used as a loading control. (f) Quantitative representation of the levels of a radio-labelled glucose analogue, 2-DOG, taken up by cells transfected with the Glut1 constructs. Note the increase in the labelled 2-DOG in cells that were transfected with the Glut1 constructs. Also note, that pAAV denotes the fact that the relevant construct was a plasmid. *, P<0.05, N≥3 assays, one-way ANOVA.