Figure 8.

Overexpression of ubiquitin restores synaptic structure in LDN-treated neurons. Cultured neurons were treated with vehicle (DMSO) or LDN for 24 h. EGFP or Myc-ubiquitin Sindbis virions were added directly to culture media after 12 h of treatment, and protein expression was allowed to continue for 12 h. At the end of treatments, neurons were fixed, permeabilized, and immunolabeled with anti-PSD-95 or colabeled with anti-PSD-95 and -Myc antibodies. A–D, Representative maximum z-projected confocal images of straightened dendrites from GFP (DMSO), GFP (+LDN), myc-ubiquitin (DMSO), and myc-ubiquitin (+LDN), respectively. E, F, PSD-95 protein puncta size (E) and number (F) were analyzed in neurons that expressed EGFP or myc-ubiquitin. The mean puncta size and number from three independent experiments were normalized to the GFP (DMSO) control. The number of puncta was calculated per 10 μm dendrite length. Measurements for PSD-95 stainings were made on >60 dendrites per condition. Scale bar, 5 μm. Mean values ± SEM are shown. *p < 0.05, **p < 0.001, unpaired Student's t test.