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. 2010 Nov 12;87(5):593–603. doi: 10.1016/j.ajhg.2010.10.001

Figure 2.

Figure 2

SCA23 Mutations Affect PDYN Processing and Enhance Dyn A Toxicity

(A) Expression and processing of WT and mutant PDYNs in RINm-5F cells. Immunoblotting was performed with anti-PDYN (against C-terminal fragment; top) or anti-Dyn B (bottom) antibodies. Synthetic big dynorphin (a 32 amino acid peptide consisting of Dyn A and Dyn B) and Dyn B were loaded on the same gel as peptide markers and produced the 4 and 2 kDa bands identified by anti-Dyn B antibodies (data not shown; however, their positions are shown on the right). The 10, 4, and 2 kDa PDYN processing products were detected in RINm-5F cells transfected with PDYN expression plasmids.

(B–D) Peptide levels in cells expressing WT PDYN were taken as 100%. One-way ANOVA followed by Dunnett's multiple comparison test; ∗∗p < 0.01, ∗∗∗p < 0.001; mutant versus wild-type. Data are shown as means ± SEM.

(B) RINm-5F cells expressing PDYN p.L211S and p.R212W showed significant elevation in levels of Dyn A compared to cells expressing WT PDYN (ANOVA; p < 0.001). The Dyn A RIA was not hindered by the mutations because they did not affect the binding of the mutant peptides to the Dyn A antibodies. The antibodies were generated against the C-terminal Dyn A fragment.12,19

(C) Dyn B levels showed significant decrease in cells expressing PDYN p.R138S, p.R212W, and p.R215C (ANOVA; p < 0.001).

(D) PDYN p.R138S and p.R215C produced lower levels of Leu-enkephalin-Arg compared to WT PDYN (ANOVA; p < 0.005). Leu-enkephalin-Arg peptides derived from PDYN p.L211S and p.R212W were not analyzed by RIA because these two mutations are located within the Leu-enkephalin-Arg antigenic epitope and may hinder the binding of these mutant peptides to anti-Leu-enkephalin-Arg antibodies.12,19

(E and F) Time-lapse imaging of striatial neurons treated with Dyn A peptides (100 nM) revealed marked loss of neuronal viability induced by Dyn A p.R212W and Dyn A p.R215C peptides compared to vehicle-treated control or WT Dyn A (one-way repeated-measures ANOVA; p < 0.005). Data are represented as the means ± SEM.

(F) Neuronal death at 60 hr induced by mutant Dyn A peptides. The level of neuronal death as a result of excess WT Dyn A was considered as 1. Bonferroni's multiple correction test, ∗p < 0.05. Data are represented as the means ± SD.