Figure 6. N-cofilin compensates the loss of ADF at synapses.

(A) Immunoblot analyses show enrichment of synaptic markers (PSD-95, synaptophysin) in synaptosomes. Equal protein load was verified by Coomassie staining of SDS-PAGEs. Compared to total protein lysates (homogenate) and the organelle- and nuclei-containing fraction P1 PSD-95 and synaptophysin signals were increased in synaptosomes. (B) Immunoblots showing that synaptic n-cofilin levels were increased in synaptosomes from ADF-KO. (C) Immunoblot analysis demonstrating equal cytosolic actin levels in CTR, n-cofilin mutants (n-Cof^{flx/flx,CaMKII-cre}), ADF-KO and double mutants (ACC) that lack both ADF and n-cofilin. Conversely, actin levels were increased in microsomal and synaptosomal preparations from ACC mice. (D) Quantification of microsomal actin levels. In microsomes from ACC mice, actin levels were significantly increased to 266.8±15.8% of CTR levels (n = 4; P<0.001) and significantly higher compared to n-Cof^{flx/flx,CaMKII-cre} mice (P<0.001) or ADF-KO (P<0.001). No significant increase in actin levels was found in microsomes from ADF-KO or n-cofilin mutants. (E) Quantification of actin levels in synaptosomes. Actin levels in synaptosomes were significantly increased in n-cofilin mutants (+10.1±2.1%; n = 4; P<0.05) and ACC mice (+27.8±5.2%; n = 4; P<0.01). In ACC mice, synaptosomal actin levels were significantly higher than in synaptosomes from n-cofilin mutants (P<0.05) or ADF-KO (P<0.01). No increase was found in synaptosomes from ADF-KO.