Figure 8. Exogenous Nmnat2 protects transected neurites for prolonged periods only when highly overexpressed.

(A) Protection of transected neurites by exogenous expression of FLAG-Nmnat2 or FLAG-Wld^S (see also Figure S9). SCG neurons injected with 1, 2.5, or 50 ng/µl empty vector (FLAG-empty) or FLAG-Nmnat2 or FLAG-Wld^S expression vectors, together with pDsRed2-N1 (50 ng/µl), were transected 48 h later. Survival of DsRed2-labeled neurites 24 h after transection is shown as a percentage of the number of labelled neurites with normal morphology just after cut (0 h) and was quantified from two to four independent experiments combining data from multiple fields (error bars = ±S.E.M). FLAG-Wld^S protects neurites significantly better than FLAG-Nmnat2 24 h after cut at the 1 ng/µl vector concentration (***p<0.001, t test). There is no significant difference in protection 24 h after transection at the 50 ng/µl vector concentration. (B) Representative fluorescent images of transected DsRed2-labeled neurites of SCG neurons injected with selected concentrations of FLAG-Nmnat2 or FLAG-Wld^S expression vectors (as indicated). The same field of neurites is shown at the time of cut (0 h) and 24 h later. The cut site is located to the left of each image. Increased magnification of the framed region in each panel is shown for better visualization of neurite morphology.