Reelin signals through phosphatidylinositol 3-kinase and Akt to control cortical development and through mTor to regulate dendritic growth

Abstract

Reelin is an extracellular matrix protein with various functions during development and in the mature brain. It activates different signaling cascades in target cells, one of which is the phosphatidylinositol 3-kinase (PI3K) pathway, which we investigated further using pathway inhibitors and in vitro brain slice and neuronal cultures. We show that the mTor (mammalian target of rapamycin)-S6K1 (S6 kinase 1) pathway is activated by Reelin and that this depends on Dab1 (Disabled-1) phosphorylation and activation of PI3K and Akt (protein kinase B). PI3K and Akt are required for the effects of Reelin on the organization of the cortical plate, but their downstream partners mTor and glycogen synthase kinase 3beta (GSK3beta) are not. On the other hand, mTor, but not GSK3beta, mediates the effects of Reelin on the growth and branching of dendrites of hippocampal neurons. In addition, PI3K fosters radial migration of cortical neurons through the intermediate zone, an effect that is independent of Reelin and Akt.

FIG. 1.

Reelin-induced phosphorylation of mTor and S6K1 depends on Dab1 phosphorylation by Src kinases. Cortical neurons from wild-type or Dab1 mutant (Scrambler) brains were cultured for 3 days and stimulated for 20 min with Reelin (+, supernatant from transfected cells) or supernatant from mock-transfected cells (−). Wild-type neurons were stimulated in the presence or absence of the Src kinases inhibitor PP2 (20 μM). (A) Cell lysates were analyzed by Western blotting to estimate the phosphorylation of the indicated proteins. (B) Comparison of mTor and S6K1 phosphorylation in Reelin- and mock-stimulated neurons. Bars are standard errors of the means from four independent experiments. **, significantly different using Student's paired t test (P < 0.01).

FIG. 2.

Reelin-induced phosphorylation of mTor and S6K1 depends on PI3K and Akt. Cortical neurons were cultured for 3 days and stimulated for 20 min with control supernatant (lanes 1, 3, 5, 7, and 9) or with Reelin (lanes 2, 4, 6, 8, and 10). Cultures were incubated with LY294002 (50 μM) (lanes 3 and 4), TCBN (1 μM) (lanes 5 and 6), rapamycin (200 nM) for 30 min (acute, lanes 7 and 8), or rapamycin (200 nM) for 24 h (long-term, lanes 9 and 10) before and during exposure to Reelin. Cell lysates were analyzed by Western blotting to estimate the phosphorylation of the indicated proteins.

FIG. 3.

CP development is perturbed following inhibition of PI3K or Akt. (A) Photomicrographs of sections stained with HE for appreciation of histological organization, immunolabeled for BrdU and Tbr1 to visualize the inside-out layering, and immunolabeled for chondroitin sulfate proteoglycan (CSPG) to monitor the splitting of the PP. The first row illustrates slices at the initiation of culture (zero days in vitro [DIV]). The following rows illustrate wild-type (W.T.) slices, reeler slices, and wild-type slices treated with LY294002 (50 μM) or TCBN (1 μM or 5 μM) and cultured for 2 days. BrdU was injected 2 h before slicing. Arrows point to BrdU- or Tbr1-positive cells within the CP and allow visualization of inside-out CP layering. VZ, ventricular zone; SP, subplate; SPP, superplate. Bar, 100 μm. (B) After 2 days in vitro, slice lysates were analyzed by Western blotting to estimate efficiency of the inhibitors, by comparing the phosphorylation of Akt and Gsk3β. LY, LY294002. The two bands for GSK3β are due to alternative splicing (51). (C) Slices prepared at E13 were cultured with LY294002 (50 μM), TCBN (1 μM or 5 μM), or control medium and subjected to a short pulse of BrdU. Numbers of BrdU-positive cells in ventricular and subventricular zones (a) and of apoptotic cells, positive for activated caspase 3 (b), are expressed as a percentage of the control value. Error bars are standard errors of the means from four experiments. NS, not significant using Student's paired t test. (D) Reelin signal detected in Western blots in supernatants from slices cultured for 2 days in vitro in the presence of the following inhibitors: LY294002 (50 μM), TCBN (5 μM), AktIV (2 μM), rapamycin (200 nM), LiCl (10 mM), TDZD8 (56 μM), and SB415286 (6 μM).

FIG. 4.

GSK3 and mTor activities are not involved in CP development. (A) E13 brain slices were cultured in control medium or in the presence of rapamycin (200 nM). After 2 days in vitro, slice lysates were analyzed by Western blotting to estimate phosphorylation of Akt, mTor, and S6K1. The presence of rapamycin had no effect on CP formation (HE stain), PP splitting (chondroitin sulfate proteoglycan [CSPG] labeling), or inside-out layering (BrdU and Tbr1 labeling). SP, subplate; VZ, ventricular zone. Bar, 100 μm. (B) Montage of HE-stained coronal sections in the forebrains of wild-type and GSK3β^−/− mutant embryos at E14. V, lateral ventricle; BF, basal forebrain; LGE and MGE, lateral and medial ganglionic eminences, respectively. Bar, 300 μm. (C) E13 brain slices were cultured in control medium or in presence of inhibitors of GSK3 (LiCl, TDZD8, and SB415286). After 2 days in vitro, slice lysates were analyzed by Western blotting to estimate phosphorylation of Tau. The presence of LiCl (10 mM), TDZD8 (56 μM), or SB415286 (6 μM) (histology is shown for SB415286 only) had no effect on CP formation (HE stain), PP splitting (CSPG labeling), or inside-out layering (BrdU and Tbr1 labeling). Bar, 100 μm.

FIG. 5.

The trophic effect of Reelin on dendrites is dependent on Src kinases, PI3K, Akt, and mTor but not on GSK3. E18 hippocampal neurons from reeler brains were cultured for 5 days in control medium or in the presence of inhibitors (PP2 at 10 μM, LY294002 at 50 μM [added 24 h after plating], TCBN at 1 μM, SB415286 at 6 μM, or rapamycin at 200 nM). The medium was complemented with Reelin or mock supernatant. (A) MAP2 immunofluorescence. (B) Comparison of MAP2-labeled dendrites was performed by measuring total dendrite length and the number of branches. Data are expressed as percentage of the mock-treated neurons in control condition. Bars are standard errors of the means based on analysis of 60 neurons in three independent experiments. Asterisks indicate a significant difference (P < 0.01) between Reelin- and mock-treated dendrites, using Student's paired t test.

FIG. 6.

PI3K inhibition decreases migration speed in wild-type and reeler slices independently from Akt. (A) Brain slices prepared at E14 were cultured in the presence of LY294002 (LY) (50 μM) or TCBN (5 μM) or in control medium and subjected to a short pulse of BrdU. Numbers of BrdU-positive cells in ventricular and subventricular zones (a), and of apoptotic cells, positive for activated caspase 3 (b), are expressed as percentages of control values. Error bars are standard errors of the means from of four experiments. NS, not significant using Student's paired t test. (B) Ventricular zone cells of E14 wild-type (WT) or reeler brains were labeled with the Cell Tracker green CMFDA compound. Slices prepared at the same coronal level were cultured in control medium or in presence of 50 μM LY294002 (LY) or 5 μM TCBN. Slices were fixed and processed for histology after the indicated times. The dotted line indicates the pial surface, and the arrows indicate the front of fluorescent cells. DIV, days in vitro. (C) Comparison of migration rates in the six situations at the different time points. Data are means ± standard errors of the means. ***, significantly different (P < 0.001) using Student's paired t test.

FIG. 7.

Schematic view of the effects of Reelin on PI3K pathways. The inhibitors used in the study are boxed in red. The sites phosphorylated under Reelin stimulation are indicated in green.