Figure 7. Elevated expression of HS is necessary to axon regrowth.
WT/SCI, WT after SCI; KO/SCI, T1KO after SCI. (a) Ext2 (green), an essential enzyme for HS sugar chain synthesis, was upregulated in and localized to neurons of T1KO mice after SCI. NeuN (red), a neuronal marker; Ext2-expressing cells (arrows). Scale bars, 20 μm. (b) Reverse transcriptase (RT)–PCR of genes encoding enzymes involved in GAG synthesis after SCI. The samples were collected from three sites in wild-type (WT) or T1KO mice: 2 mm rostral from the lesion centre (left), the lesion centre (centre) and 2 mm caudal from the lesion centre (right). The samples were analysed using RT–PCR. Notably, the rostral site was mainly composed of neurons, not of reactive astrocytes (the left-most cartoon). The average expression of each gene in intact WT or in intact T1KO mice was defined as 1.0. Expression of each gene (Csgalnact2 (T2), Ext1, Ext2 or Extl2) in TIKO versus that in WT. *P<0.05 (n=6; Bonferroni’s comparison test). (c) Overexpression of Ext1/Ext2 promoted axon outgrowth of WT cortical neurons in an HSase-dependent manner. Both Ext1 and Ext2 complementary DNAs (Ext1 and 2) were transfected into cultured neurons. The Ext1 and Ext2 proteins are thought to form a heterodimeric complex during HS synthesis; overexpression of Ext1 alone did not result in a significant change. *P<0.05 (one-way ANOVA; n=4); Ext1&2 versus control (GFP) or versus Ext1&2+HSase. In each individual experiment, 100 neurons were counted and the experiments were performed at least three times. Data are expressed as the mean±s.e.m. in b and c.