Figure S3.

Nrx HS Modification Does Not Affect Nrx Surface Trafficking or Neuron Survival, and Heparinase Reduces Presynaptic Differentiation, Related to Figures 2 and 3

(A) Nrx1β^∗ΔHS reaches the axon surface, like Nrx1β^∗, as shown by surface labeling of the V5 tagged Nrx constructs co-expressed with YFP through a 2A peptide. Note that both Nrx1β^∗ and the ΔHS mutant are primarily axonal as MAP2 positive dendrites show little V5 fluorescence.

(B) The axon surface level of Nrx1β^∗ΔHS was indistinguishable from that of Nrx1β^∗. Ratios of surface V5 to total YFP were assessed along axons of 13 DIV neurons co-expressing the V5 tagged Nrx constructs with YFP linked through the 2A peptide. A Mann-Whitney test showed no significant difference, n = 21-22 cells from 2 independent experiments.

(C) mEPSC amplitude was not significantly different among groups (p = 0.0591). The numbers of cells were: CFP+shCon (n = 21); CFP+shNrx (n = 32); Nrx^∗+shNrx (n = 29); Nrx^∗ΔHS+shNrx (n = 32) from 4 independent experiments. mIPSC amplitude was reduced by Nrx knockdown and partially rescued by RNAi-resistant Nrx^∗ but not Nrx^∗ΔHS. The numbers of cells were: CFP+shCon (n = 20); CFP+shNrx (n = 18); Nrx^∗+shNrx (n = 18); Nrx^∗ΔHS+shNrx (n = 18) from 3 independent experiments. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 by Kruskal-Wallis and Dunn’s tests compared to shCon+CFP. For the experiments shown here and in Figure 2, neurons were transfected at plating with the rescue constructs, exposed to knockdown AAV vectors from 3 DIV and analyzed at 13 DIV (mIPSCs) or 14 DIV (mEPSCs).

(D and E) Hippocampal neuron cultures immunolabeled for MAP2-positive dendrites and tau-positive axons appeared similar among all groups. Neuron density assessed as number of MAP2-positive neurons per coverslip area normalized to the shCon+CFP group did not differ among groups by Kruskal-Wallis, n = 30 from 3 independent experiments. Neurons analyzed here were from the same experiments as for Figures 2, 3D-3E and panel C in this figure. Neurons were transfected at plating with the rescue constructs, exposed to knockdown AAV vectors from 3 DIV and analyzed at 14 DIV.

(F) Clustering of presynaptic marker bassoon in contacting axons induced by NL1, NL2, or LRRTM2 on COS7 cells, but not by NGL-3 a ligand of type IIA protein tyrosine phosphatases, was reduced by heparinase (Heps). Co-labeling for the microtubule associated protein 2 (MAP2) identified dendrites to exclude native synapses from the analysis. Heparinase was added after axon outgrowth only during the co-culture period. For quantitation, see Figure 3C.

Error bars represent SEM. Scale bars, (A) 10 μm, (E) 10 μm, (F) 100 μm.