Figure 1.
NRP1 and NRP2 are expressed in adult neural progenitor cells and can be knocked down by NRP1 and NRP2 shRNA respectively.
(A) Representative western blots showing that NRP1 and NRP2 are expressed in cortical and hippocampal primary neuronal culture and adult hippocampal neural progenitor cells (NPCs). HEK 293 cells were overexpressed with NRP1 (‘+’, upper panel) and NRP2 (‘+’, lower panel) and probed with anti-NRP1 and anti-NRP2 respectively. Untransfected HEK 293 cell lysate (‘−’, upper and lower panels). (B) A schematic diagram of retroviral constructs co-expressing GFP under EF1-α promoter and shRNA driven by the U6 promoter. (C) Retroviral constructs expressing different shRNAs were co-transfected with the indicated myc-tagged overexpression constructs into 293T cells. Western Blot analysis showed effective knockdown of wild-type NRP1 and NRP2 by shRNAs for NRP1 and NRP2. pcDNA3.1-nrp1-782 and pcDNA3.1-nrp1-2231 express NRP1 that harbor silent mutations rendering them are resistant to knockdown by pUEG-nrp1-782 and pUEG-nrp1-2231, respectively. pcDNA3.1-nrp2-238 and pcDNA3.1-nrp2-1076 are resistant to knockdown by pUEG-nrp2-238 and pUEG-nrp2-1076, respectively.
Figure 2.
NRP1 and NRP2 regulated dendritic morphology of newborn DGCs in adult brain.
(A) Representative images and tracings of dendrites of control, NRP1 or NRP2-shRNA expressing DGCs at 14 dpi (Scale bar, 20 µm). (B, C) Quantification of total dendritic length and branch number of newborn DGCs. Each symbol represents a single DGC. (**P<0.01, 1-way ANOVA with Newman-Keuls’ post hoc test). (D) Graph showing dendritic complexity of GFP+ DGCs. Values represent mean ± s.e.m. (**P<0.01, *P<0.05, 1-way ANOVA with Newman-Keuls’ post hoc test).
Figure 3.
Overexpression of shRNA-resistant NRP1 and NRP2 rescues phenotype induced by NRP1 and NRP2 shRNA.
Mixture of retrovirus expressing the indicated constructs was injected into dentate gyrus. Quantification of total dendritic length (A) and branch number (B) of newborn DGCs at 14 DPI. 30–50 neurons from each group were used for analysis. (**P<0.01, 1-way ANOVA with Newman-Keuls’ post-hoc test). NRP1*: NRP1 shRNA-resistant NRP1, NRP2*: NRP2 shRNA-resistant NRP2.
Figure 4.
Single cell knockout of NRP1 in neural progenitors results in neurons with impaired dendritic formations is independent of vascular endothelial growth factor (VEGF)-binding of NRP1.
(A) Graphs showing quantification of total dendritic length (A) and branch number (B) of newborn DGCs expressing GFP (CTR) or Cre-recombinase (CRE) at 14 DPI in NRP1 conditional knockout mice (NRP1 Floxed/Floxed) or mice expressing one single copy of Floxed-NRP1 and a knockin (KI) of an altered ligand binding site variant of Nrp1 (NRP1 Floxed/KI). (C) Representative tracings of dendritic morphology of adult-born neurons from the above indicated groups. (Scale bar = 20 µm).
Figure 5.
Semaphorin 3A/3F increased dendritic growth in primary neurons and induce tyrosine and serine phosphorylation of FAK.
(A) Representative images showing neurons treated with AP-control (AP), Sema 3A-AP (3A) and 3F-AP (3F). Scale bar = 20 µm. (B, C and D) Graphs showing total dendritic length (B), total number of dendritic branches (C) and axon length (D) of primary hippocampal neurons treated with AP control, Sema3A or Sema 3F. (E, F and G) Representative blots showing phosphorylation of FAK in primary neurons (E and G) and 293T cells (F) treated with AP control, Sema3A or 3F. Graph displays change relative to total FAK and values are mean ± s.e.m, n = 3 independent experiments. 40–50 neurons from each group were used for analysis. Values represent mean ± s.e.m. (**P<0.01, 1-way ANOVA with Newman-Keuls’ post-hoc test).
Figure 6.
Expression of FAK phosphorylation-defective mutants in primary neurons impaired semaphorin-induced dendritic growth.
(A) Representative images of primary hippocampal neurons transfected with vector, wild-type FAK (WT), Y397F FAK (Y397F), S732A FAK (S732A) and Y397F/S732A FAK (Y397F/S732A). Neurons were treated with AP control (AP), 3A-AP (3A) or 3F-AP (3F). Scale bar = 20 µm. (B) HEK293T cells transfected with the indicated constructs encoding FAK or its mutants validating the phosphorylation status of these constructs. (C, D and E) Graphs showing percentage dendritic growth of primary hippocampal neurons expressing FAK mutant constructs as indicated, treated with AP control (C), Sema3A (D) or 3F (E), with percentages normalized to Vector+AP. 30–100 neurons from each group were used for analysis. Values represent mean ± s.e.m. (**P<0.01, 1-way ANOVA with Newman-Keuls’ post-hoc test).
Figure 7.
Cdk5 was required for regulating dendritic morphology of primary neurons.
(A) Representative blots showing phosphorylation of Cdk5 in HEK293T cells transfected with NRP1 or 2 as indicated, treated with AP control, Sema3A or 3F. (B) Representative blots showing phosphorylation of FAK, paxillin and DCX in primary hippocampal neurons pretreated with vehicle (−R) or roscovitine (+R) prior to AP-control, Sema3A or 3F treatment. Graphs displays changes relative to FAK and values are mean ± s.e.m, n = 3 independent experiments. (C) Graph showing percentage dendritic growth of primary hippocampal neurons treated with vehicle (−R) or roscovitine (+R) together with AP, Sema3A or 3F. 50 neurons from each group were used for analysis. Values represent mean ± s.e.m. (**P<0.01, 2-way ANOVA with Bonferroni’s post-hoc test).
Figure 8.
Roscovitine inhibition of Sema3A or 3F-induced dendritic growth was rescued by overexpression of wild-type FAK.
(A) Representative images of primary hippocampal neurons transfected with vector or wild-type FAK (WT). Neurons were treated with AP control (AP), 3A-AP (3A), 3F-AP (3F), AP with roscovitine pre-treatment (AP+R), 3A-AP or 3F-AP with roscovitine pre-treatment (3A+R). Scale bar = 20 µm. (B) Representative blots showing phosphorylation of FAK in primary hippocampal neurons transfected with vector or WT-FAK treated with AP, Sema3A or Sema 3F together with vehicle (−R) or roscovitine (+R). (C, D and E) Graphs showing total dendritic length of primary hippocampal neurons expressing GFP (vector) or WT-FAK and treated with vehicle or roscovitine together with AP control (C), Sema3A (D) or 3F (E). 30–100 neurons from each group were used for analysis. Values represent mean ± s.e.m. (**P<0.01, 2-way ANOVA with Bonferroni’s post-hoc test).
Figure 9.
Overexpression of constitutively active FAK rescues the effects of roscovitine inhibition of semaphorin-induced dendritic growth.
(A) Representative images of primary hippocampal neurons transfected with constitutively active FAK (S732D). Neurons were treated with AP control (AP), 3A-AP (3A), 3F-AP (3F), AP with roscovitine pre-treatment (AP+R), 3A-AP or 3F-AP with roscovitine pre-treatment (3A+R). Scale bar = 20 µm. (B) Graphs showing total dendritic length of primary hippocampal neurons expressing S732D FAK treated as indicated above. 40–50 neurons from each group were used for analysis. Values represent mean ± s.e.m. (No significant difference by 2-way ANOVA).
Figure 10.
Knockdown of downstream mediators of semaphorin signaling, Cdk5 and FAK, induced defective dendritic morphologies of newborn DGCs in adult brain.
(A) Representative images and tracings of dendrites of control, Cdk5 or FAK-shRNA expressing DGCs at 14 dpi (Scale bar, 20 µm). Graphs showing quantification of total dendritic length (B) and branch number (C) of newborn DGCs. Each symbol represents a single DGC at 14 dpi. (**P<0.01, 1-way ANOVA with Newman-Keuls’ post-hoc test).
Figure 11.
Overexpression of Cdk5 or FAK rescued dendritic phenotypes of NRP-1 and -2 deficient neurons.
(A) Representative images of adult-born neurons co-infected with retrovirus expressing shNRP1 and mCherry shNRP1 and mCherry-T2A-Cdk5 and shNRP1 and mCherry-T2A-FAK. Scale bar = 20 µm. Quantification of total dendritic length (B, D) and branch number (C, E) of newborn DGCs at 14 dpi. Each symbol represents data from a single DGC at 14 dpi. (**P<0.01, 1-way ANOVA with Newman-Keuls’ post hoc test).