Figure 1
From: Heparanase attenuates axon degeneration following sciatic nerve transection
Microarray, qPCR and protein analysis indicates a role for HS mediated signalling in Wallerian degeneration. (A) Heat map to show microarray analysis from two different published array sets obtained from GEO (refer to methods) for HSPG genes as well as genes associated with HS biosynthesis and regulation. Genes coloured yellow are not differentially expressed (DE) while genes with increasing expression become a gradually darker green, conversely genes that decrease in expression become gradually darker in orange and then red. The dendrogram, which illustrates clustering between genes, used a Euclidean measure to calculate distance and the complete agglomeration method for clustering, according to the heatmap.2 R package. Clustering of Ctsl, Hpse and Gpc1 is indicated by the box. Vertical bold line separates the profile from WT (left) and Wlds (right) mice. D = day. (B) RT-qPCR validation of selected highly expressed genes from the microarray results using an ex vivo SN injury model confirms Hpse is highly (significantly) up-regulated. All results compare uninjured SN to ex vivo SN after either 2 or 4 DIV. Hpse (2 DIV - *p = 0.03, n = 5, 4 DIV – **p = 0.0009, n = 6). Ctsl (2 DIV - p = 0.2, n = 3, 4 DIV – p = 0.12, n = 3). GALNS (2 DIV – *p = 0.043, n = 3, 4 DIV – *p = 0.006, n = 6). E) Gpc1 (2 DIV - *p = 0.06, n = 4, 4 DIV – p = 0.22, n = 3) Gpc6 (2 DIV – *p = 0.04, n = 4, 4 DIV = p = 0.059 n = 3). (C) Representative full Western blot from one experiment of, ex vivo SN, illustrates an upregulation in immunoreactivity for 3G10 antibody, that detects HS cleaved by Hpse, after 2 DIV (injured) compared to uninjured control – where no immunoreactivity was detected (n = 3). Amido black whole protein stain was used for loading control (refer to supplementary section Fig. S4). (D) Immunoreactivity/fluorescent staining for the antibody IOE4, which recognises a wide array of HS expression, demonstrates baseline presence of HS in the uninjured SN. Scale bar = 50 μM. (E) Immunostaining for Sox2 and DAPI in purified rat Schwann cells expressing endogenous GFP shows that (F) Hpse is expressed in purified rat Schwann cells using PCR (n = 2). Students T test or one sample T test was used for all statistical analysis.
