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First published online 13 May 2008
doi: 10.1242/jcs.027334

Journal of Cell Science 121, 1869-1875 (2008)
Published by The Company of Biologists 2008
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Reduction of Crk and CrkL expression blocks reelin-induced dendritogenesis

Tohru Matsuki, Albéna Pramatarova and Brian W. Howell*

Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, NIH, 35 Convent Drive, Bethesda, MD 20892, USA


Figure 1
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  		Fig. 1. The complexity of pyramidal neurons in the CA1 region is reduced by excision of a Dab1 expression cassette in postnatal mice. Mice that were homozygous for the Dab1 cKI allele (A-E) or homozygous for the Dab1 cKI allele and carried a tamoxifen-inducible Cre transgene (Cre-ERTM; F-J) were injected with tamoxifen at P3 and analyzed for Dab1 and β-galactosidase expression (βG) (B,G), Nuclear Fast Red (FR) and β-galactosidase activity (C,H) at P7, or analyzed by Golgi staining at P60 (D,I). Hematoxylin staining of the hippocampus shows that neuronal cell body position is relatively normal in Dab1 cKI homozygous animals in the absence or presence of the Cre-ERTM transgene at P7 (A,F, respectively) and P60 (E,J, respectively) in tamoxifen-treated animals. Pyramidal neurons from the CA1 region of the hippocampus of adult animals have the basic morphology characterized in the diagram. Golgi-stained neurons from the CA1 region of the hippocampus were analyzed in two control groups of mice that did not carry the Cre-ERTM gene but were treated with tamoxifen (K,L), and mice that carried the Cre-ERTM gene but were not treated with tamoxifen (M,N), as well as one group of experimental animals that carried the Cre-ERTM transgene and were treated with tamoxifen (O,P). The neuronal morphologies displayed are representative of those observed in several sections of at least three brains from each treatment group. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum moleculare. Bar, 500 µm in G,J and 20 µm in H,P.

 

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  		Fig. 2. Inhibitory RNAs directed against Crk and CrkL effectively reduce expression of their respective targets. (A) The protein expression of Crk and CrkL was compared between cultures of B16 mouse melanoma cells (lanes 1-4) or primary neurons (lanes 5-7) in the absence of infection (lane 1), after infection with control virus (lanes 2, 5), virus targeting Crk&CrkL (lanes 3, 6) or a mutant version of the shRNA (mut-shRNA, lanes 4, 7). Similarly, the effects of the CrkL shRNA lentivirus on Crk and CrkL expression were compared in uninfected (lane 8) and control infected (lane 9) B16 cells, and in those infected with CrkL shRNA virus (lane 10) and mutant CrkL shRNA virus (lane 11) by western blotting. (B-D) The intensity of bands on western blots was averaged between three independent experiments. (B,C) The Crk&CrkL shRNA reduced expression of CrkI and CrkII in B16 cells, and in neurons a significant reduction of CrkL was also observed. (D) The CrkL shRNA reduced expression of CrkL and CrkI in B16 cells, but had no effect on CrkII.

 

Figure 3
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  		Fig. 3. Reduction of Crk and CrkL prevents reelin-enhanced dendrite extension in hippocampal neurons. Primary hippocampal neurons harvested from E17 mouse brains were infected with lentiviruses. Two days later, neurons were replated in neurobasal/B-27 growth media (A,D,G), control conditioned media (CM) (B,E,H) or reelin CM (C,F,I). Map2-positive dendrites of neurons infected with GFP control virus, measured after 5 days of growth, were longer when grown in reelin CM (C) than control CM (B) or neurobasal media (A). Reelin CM treatment also enhanced dendritogenesis of neurons infected with CrkL shRNA virus (F) as compared with control CM (E) or neurobasal media (D) treatment of the same population of neurons. By contrast, reelin CM treatment did not promote extension of neurons infected with Crk&CrkL shRNA virus (I) relative to control CM (H) or neurobasal (G) treatments. Bar, 50 µm. (J) Quantification of these and similar experiments demonstrated that reelin CM promoted an ~twofold increase in dendrite length that was blocked by Crk&CrkL shRNA virus infection, but not by Crk&CrkL mutant (mt) shRNA, or GFP-expressing control viruses. SFK inhibitors PP1 and PP2 also prevented reelin CM-enhanced dendritogenesis. The combined effect of SFK inhibitors and Crk&CrkL shRNA viruses did not cause further reductions in process lengths over that induced by either agent alone. The CrkL shRNA virus did not significantly inhibit reelin CM-enhanced dendritogenesis. The number of neurons measured is indicated at the base of each bar. *P<0.001. Error bars indicate s.e.m.

 

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  		Fig. 4. BDNF-enhanced hippocampal dendrite outgrowth is blocked by Src inhibitors but not by the Crk&CrkL shRNA virus. BDNF enhances the growth of Map2-positive hippocampal dendrites ~1.8-fold after 5 days of treatment, as compared with growth in neurobasal/B-27 media alone. Inclusion of the SFK family kinase inhibitors PP1 and PP2 during incubation prevented the BDNF-induced augmentation of process extension. However, reduction of Crk and CrkL expression did not significantly block the effect of BDNF on dendrites. The number of neurons measured is indicated at the base of each bar. *P<0.001. Error bars indicate s.e.m.

 

Figure 5
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  		Fig. 5. Axon lengths of hippocampal neurons are not altered by reelin treatment or by reduction in Crk levels. Tau-positive axons lengths were measured in hippocampal neurons 3 days after viral infection and 1 day after replating in the presence of reelin CM, control CM or neurobasal/B-27 media. Reelin CM treatment did not significantly alter process lengths under these conditions. Similarly, infection of neurons with the Crk&CrkL shRNA virus had no effect on the extension of axons in hippocampal neurons. The number of neurons measured is indicated at the base of each bar.

 

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