Fig. 7

Smcx Is Required for Dendritic Development in Rat Cerebellar Granule Neuron

(A) Representative images of primary cultured rat cerebellar granule neurons, which were cotransfected with the U6/scramble, U6/smcxA, or U6/smcxB shRNA plasmid and a GFP expression vector and subjected to immunocytochemistry with a GFP antibody. Arrows and arrowheads indicate dendrites and axons, respectively. Scale bar denotes 50 μm.

(B) Analysis of dendritic length of neurons transfected in (A) shows that knockdown of Smcx led to a significant decrease in total dendritic length with two different RNA hairpins (p < 0.0001; ANOVA followed by Bonferonni-Dunn posthoc test, 160 neurons measured). Data presented as mean ± SEM.

(C) SMCX-dependent dendritic morphogenesis is linked to SMCX demethylase activity. Primary granule neurons were transfected with U6/scramble or U6/smcx B RNAi plasmid, together with a control expression vector or an expression plasmid encoding the RNAi-resistant human SMCX protein (WT Res) or point mutants that disrupt demethylase activity (H514A Res and F642L Res). Dendritic length was significantly reduced in Smcx knockdown neurons as compared to U6/scramble-transfected neurons (p < 0.0001; ANOVA followed by Bonferonni-Dunn posthoc test). Dendritic length was significantly increased in neurons expressing WT Res SMCX, but not in H514A Res- or F642L Res-expressing neurons, in the background of Smcx RNAi, as compared to Smcx RNAi alone (p < 0.0041; ANOVA followed by Bonferonni-Dunn posthoc test). Total number of neurons measured was 265. Data presented as mean ± SEM.