1 Nature Neuroscience 2006 Vol: 9(8):993-995. DOI: 10.1038/nn1742

IGF-1 receptor is essential for the establishment of hippocampal neuronal polarity

How a neuron becomes polarized remains largely unknown. Results obtained with a function-blocking antibody and an siRNA targeting the insulin-like growth factor-1 (IGF-1) receptor suggest that an essential step in the establishment of hippocampal neuronal polarity and the initiation of axonal outgrowth is the activation of the phosphatidylinositol 3-kinase (PI3k)-Cdc42 pathway by the IGF-1 receptor, but not by the TrkA or TrkB receptors.

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Figures
Figure 1: Reducing IGF-1R function by incubation with blocking antibody or by siRNA silencing prevents axon formation in rat hippocampal neurons.(a) Double immunofluorescence micrographs showing the distribution of tyrosinated tubulin and active IGF-1 receptor in hippocampal pyramidal neurons after 12 h (top) or 18 h (bottom) in culture (Supplementary Methods). (b) Micrographs of hippocampal pyramidal neurons immunostained with antibody to tyrosinated tubulin in control cultures (Co) and cultures grown in the presence of an antibody blocking the IGF-1R (Ab). (c) Percentage ( s.e.m.) of neurons at specific stages of differentiation, grown for 24 h or 48 h in culture, in control conditions (Co) or in the presence of an IGF-1R–blocking antibody (Ab). n = 3 independent experiments. At least 150 cells were scored for each condition. (d) Western blots showing protein levels of gc, TrkB and Cdc42 in control cells (Co) and cells transfected with a scrambled RNA sequence (ss) or IGF-1R siRNA (si). Transfection efficiency was 60–70%. A substantial decrease of gc expression in the siRNA-treated cells is evident. (e) Triple immunofluorescence micrographs showing the distribution of gc, tyrosinated -tubulin and IGF-1R–targeted, biotinylated siRNA in hippocampal pyramidal neurons cultured for 24 h in the presence of 50 ng ml-1 BDNF. The transfected neuron (arrow) did not develop an axon. (f) Percentage ( s.e.m.) of control neurons (ssRNA) or neurons containing IGF-1R–targeted siRNA at specific stages of differentiation after 24 h in culture in the presence of 50 ng ml-1 BDNF or 50 ng ml-1 BDNF + 50 ng ml-1 NGF. n = 3 independent experiments. At least 150 cells were scored for each condition. Scale bars: 20 m in a and e; 60 m in b. Figure 2: Cotransfection of neurons with IGF-1R–targeted siRNA and cDNA encoding fc- Cdc-42 rescues the phenotype.(a,b) Triple immunofluorescence micrographs showing the distribution of tyrosinated -tubulin, IGF-1R siRNA and either (a) active PI3k (p-tyr-p85) or (b) mPar3 in hippocampal pyramidal neurons cultured for 24 h in the presence of 50 ng ml-1 BDNF. (c) Triple immunofluorescence micrographs showing the distribution of Cdc42, tyrosinated -tubulin and gc in hippocampal pyramidal neurons (24 h in culture in the presence of 50 ng ml-1 BDNF). The transfection efficiency of the cells shown in panels a–c was 60–70%. All cells cultured in the presence of ssRNA in stage 3 (72 4%) showed polarized distributions of active PI3k, mPar3 and Cdc42. In contrast, no cells transfected with the siRNA in stage 2 showed polarization of these antigens. (d) Western blots of GTP-bound Cdc42 from neurons transfected with ssRNA or siRNA. Numbers below are the average optical densities s.e.m. of three independent experiments. (e) Triple immunofluorescence micrographs of hippocampal pyramidal neurons cotransfected with the DNA encoding fc-Cdc42 and IGF-1R siRNA (top) or transfected only with siRNA (bottom), and grown for 24 h in vitro. Scale bar, 15 m.
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References
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    • . . . A widely used model to elucidate the underlying molecular mechanisms is the primary dissociated hippocampal neuron of the rat in culture1 . . .
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    • . . . In these cells, accumulation of active phosphatidylinositol 3-kinase (PI3k) and its product PIP3 at the growth cone of an undifferentiated neurite is crucial for the outgrowth of the future axon and the polarized distribution of mPar3 and mPar6, two proteins required for axon specification2, 3, 4 . . .
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    • . . . By contrast, IGF-1R knockout mice have serious defects in CNS development, including a marked decrease in the number of axons11 . . .
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    • . . . Notably, TrkB-null mice show no obvious structural abnormalities in the CNS (ref. 15) . . .
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