Fig. 4

KD/KO of Pin1 Reduces Axon Growth in Primary Neurons and Is Fully Rescued by CRMP2A Overexpression

Primary cortical neuron cultures were derived from embryos of three independent Pin1 WT (A-F) and KO (G-L) mouse littermates and infected with lentiviruses expressing control vector (A and G), FLAG-Pin1 (B and H), Sh-CRMP2A (C and I), Sh-Pin1 (D and J), or non-silencing shRNA (NSC) (E and K) lentiviruses. Their axon length was determined at 3 DIV by immunostaining for the axon marker tau (green) and the dendrite marker MAP2 (yellow). Axon tracings are shown in violet. (F) and (L) show the means ± SEM values calculated from quantification of at least two optical fields and at least 50 neurons in each experiment. In Pin1 WT neurons, while overexpression of Pin1 had no significant effect, KD of CRMP2A or Pin1 in Pin1 WT neurons significantly reduced axon length (**p < 0.0001). In Pin1 KO neurons, overexpression of Pin1 completely rescued axon length, but KD of CRMP2A or Pin1 did not significantly reduce axon length, although Sh-CRMP2A neurons had slightly shorter axon. (M-P) CRMP2A overexpression fully rescues shortened axon length in Pin1 KO neurons. Pin1 WT (M and O) and KO (N and P) primary cortical neurons were co-transfected with GFP and vector control (M and N) or GFP and FLAG-CRMP2A (O and P) and axon growth was analyzed at 7 DIV in GFP-positive neurons. Outlines of the neurons are shown in lower right boxes. Upper right panel indicates quantification of the axon lengths as mean ± SEM (*p < 0.05, **p < 0.001). Scale bars, 100 µm. (M, O, and P) are composite images of four, six, and seven images, respectively. See also Figure S3.