Figure 2. Knockdown of α-actinin-2 increases spine number and inhibits spine maturation.

A) Hippocampal neurons were co-transfected at DIV 17 with GFP and either pSUPER, pSUPER-α-actinin-2, or pSUPER-α-actinin-2 plus α-actinin-2-SS. Neurons were fixed on DIV 21 and scored for (B–D) changes in spine density, head width, and morphology. Inhibition of α-actinin-2 at DIV 17 increases spine density, B. For each condition, spine density was analyzed on 35–42 dendrites from 3 separate cultures. The fraction of spine head widths >0.4 µm is significantly reduced in neurons with α-actinin-2 knocked down at DIV 17, C. α-Actinin-2 knockdown creates an increase in the fraction of filopodia-like spines and a decrease in the fraction of mushroom-shaped spines, thin spines (long protrusions with small head at tip), and stubby spines, D. For quantification of spine head width and morphology, 1444–2081 spines from 51 control neurons, 40 α-actinin-2 knockdown neurons, and 35 rescue neurons of 3 separate cultures were analyzed. E) Hippocampal neurons were transfected at DIV 6 with either GFP alone or GFP + α-actinin-2-SS. Neurons were fixed on DIV 22 and scored for (F–H) changes in spine length, head width, and morphology. Overexpression of α-actinin-2-SS increases spine length (F) and reduces spine head width (G). α-Actinin-2-SS overexpression creates an increase in the fraction of filopodia-like spines and a decrease in the fraction of stubby spines, H. For quantification of spine length, head width, and morphology, 311–610 spines from 15 control neurons and 21 α-actinin-2-SS overexpression neurons of 3 separate cultures were analyzed. Error bars represent SEM. p-values were derived using the paired t-test. Scale = 5 µm.