Reelin modulates NMDA receptor activity in cortical neurons - PubMed (original) (raw)

Reelin modulates NMDA receptor activity in cortical neurons

Ying Chen et al. J Neurosci. 2005.

Abstract

Reelin, a large protein that regulates neuronal migration during embryonic development, activates a conserved signaling pathway that requires its receptors, very low-density lipoprotein receptor and apolipoprotein E receptor 2, the cytoplasmic adaptor protein Disabled-1 (Dab1), and Src family kinases (SFK). Reelin also markedly enhances long-term potentiation in the adult hippocampus, suggesting that this developmental signaling pathway can physiologically modulate learning and behavior. Here, we show that Reelin can regulate NMDA-type glutamate receptor activity through a mechanism that requires SFKs and Dab1. Reelin mediates tyrosine phosphorylation of and potentiates calcium influx through NMDA receptors in primary wild-type cortical neurons but not in Dab1 knock-out neurons or in cells in which Reelin binding to its receptors is blocked by a receptor antagonist. Inhibition of SFK abolishes Reelin-induced and glutamate-dependent enhancement of calcium influx. We also show that Reelin-induced augmentation of Ca2+ entry through NMDA receptors increases phosphorylation and nuclear translocation of the transcription factor cAMP-response element binding protein. Thus, Reelin may physiologically modulate learning and memory by modulating NMDA receptor functions.

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Figures

Figure 1.

Figure 1.

Representative images of Reelin-induced enhancement of Ca2+ influx in cortical neurons. Representative images of fura-2 AM 340/380 ratios of Ca2+ transients induced by 40 μ

m

glutamate in cortical neurons are shown. A pseudocolored calibration scale for 340/380 ratios is shown on the right. All recordings were performed in ACSF supplemented with TTX, nimodipine, and CNQX. Wild-type neurons were recorded without (first row) and with (second row) Reelin treatment. Fura-2 AM 340/380 ratio images are shown 30 s before (first column) and 30 s after (second column), 1.5 min after (third column), and 3 min after (fourth column) application of 40 μ

m

glutamate.

Figure 2.

Figure 2.

Reelin enhances glutamate-stimulated Ca2+ influx through the NMDA receptor in wild-type cortical neurons. At 12–13 DIV, the cultured cortical neurons were stimulated with glutamate in the presence of TTX, nimodipine, and CNQX. A, Basal and peak 340/380 ratios are shown for individual cortical neurons. Basal ratios were determined as the average of the ratio values 35–15 s before glutamate application. Peak values were determined from maximal signals observed 25–60 s after glutamate application. Trace of 340/380 ratio for the representative cell is shown in B. Presence of glutamate in the buffer is indicated by the horizontal bar. Changes in 340/380 ratios at peak and basal were calculated for each cell as R(peak) – R(basal). Cells were categorized by their R(peak) – R(basal) values, and responding distribution of the cells was plotted in C (n = number of cells). Basal and peak 340/380 ratio, trace of representative 340/380 ratio, and distribution of Reelin responding cells are shown in D–F respectively. Basal and peak 340/380 ratios are shown for individual Reel in-treated cortical neurons perfused with Ca2+-free ACSF (G) and

d

-APV (J). Traces of 340/380 ratios for representative cells are shown in H and K. Presence of glutamate in the buffer is indicated by the horizontal bar. Responding distributions for each different treatment are shown in I and L (n = number of cells).

Figure 3.

Figure 3.

RAP, a ligand for LDL receptor family members, blocks Reelin-mediated enhancement of glutamate-stimulated Ca2+ influx. Basal and peak 340/380 ratios are shown for individual cortical neurons treated with mock media (A), GST and Reelin (D), and GST–RAP and Reelin (G). Traces of 340/380 ratios for representative cells are shown in B, E, and H. Presence of glutamate in the buffer is indicated by the horizontal bar. Responding distributions for each different treatment are shown in C, F, and I (n = number of cells).

Figure 4.

Figure 4.

Dab1 is required for Reelin-induced enhancement of Ca2+ influx. Basal and peak 340/380 ratios are shown for individual Dab1+/+ cortical neurons without treatment (A), Dab1+/+ neurons treated with Reelin (D), Dab1–/– neurons without treatment (G), and Dab1–/– neurons treated with Reelin (J). Traces of 340/380 ratio for representative cells are shown in B, E, H, and K. Presence of glutamate in the buffer is indicated by the horizontal bar. Responding distributions for each different treatment are shown in C, F, I, and L (n = number of cells).

Figure 5.

Figure 5.

Src family tyrosine kinases are required for Reelin-induced enhancement of Ca2+ influx. Basal and peak 340/380 ratios are shown for individual cortical neurons treated with PP2 (A), PP2 and Reelin (D), PP3 (G), and PP3 and Reelin (J). Traces of 340/380 ratio for representative cells are shown in B, E, H, and K. Presence of glutamate in the buffer is indicated by the horizontal bar. Responding distributions for each different treatment are shown in C, F, I, and L (n = number of cells).

Figure 6.

Figure 6.

Stimulation of NR2B tyrosine phosphorylation. A, HEK 293 cells were transfected with the indicated expression vectors, and whole-cell lysates (WCL) were analyzed by immunoblotting for SFK tyrosine-phosphorylated at Y416, total cellular Src, and Dab1. Immunoprecipitated (IP) myc-tagged NR2B was probed for tyrosine phosphorylation [pY (4G10)]. Total amounts of immunoprecipitated NR2B are shown below (NR2B). B, Cortical neuronal cultures were treated in the absence (M) or presence (R) of Reelin at 4°C for the indicated times. NR2B was immunoprecipitated from the lysates and analyzed for tyrosine phosphorylation [pY (4G10)]. Total amounts of immunoprecipitated NR2B are shown below. Densitometric scanning of a total of 16 sample lanes from three independent experiments revealed a 20-fold average increase in detectable NR2B phosphotyrosine levels in response to Reelin (SEM ± 10; p < 0.05; Student's paired t test). Values were normalized to total NR2B levels.

Figure 7.

Figure 7.

Reelin potentiates synaptically evoked NMDA currents. A, Responses were recorded using whole-cell voltage clamp at potentials varying between –70 and +70 mV. I–V curves of residual NMDA currents recorded in the presence of GABAA and AMPA receptor antagonists, with (triangles) or without (circles) nimodipine, are shown in either the presence (open symbols) or absence (filled symbols) of Reelin. Values were obtained from seven (circles) and four (triangles) different CA1 pyramidal neurons, respectively. I–V curves were recorded 10 min after Reelin application. B, Representative responses recorded at holding potentials between –70 and +70 mV (steps of 10 mV) after application of either Reelin or control fractions in the absence and presence of nimodipine. Reelin treatment significantly increased the amplitudes of NMDA currents over the entire voltage range.

Figure 8.

Figure 8.

Reelin increases glutamate-induced CREB Ser133 phosphorylation. A, Primary cortical neurons at 7 DIV were treated in the absence (M) or presence (R) of Reelin in ACSF supplemented with TTX, nimodipine, and CNQX.

d

-APV (100 μ

m

) was applied to block NMDA receptors. Cells were stimulated with 10 μ

m

glutamate for 8 min, and lysates were analyzed by Western blotting for levels of CREB, phosphorylated at Ser133(pCREB; top), total CREB (middle), and tyrosine-phosphorylated Dab1 (pDab1; bottom). The latter serves as an indicator of activation of the Reelin signaling pathway. KCl (55 m

m

) was applied in the absence of inhibitors as a positive control to depolarize neurons and induce calcium entry through voltage-gated channels. Densitometric scanning of a total of 15 sample lanes from three independent experiments revealed a statistically significant (p < 10–7) average 2.3 ± 0.14-fold increase in phospho-CREB levels in response to Reelin and glutamate compared with glutamate alone. The average increase in Reelin treatment over mock treatment in the absence of glutamate was sevenfold (SEM ± 2; p < 0.01). The density of phospho-CREB signals was normalized to the corresponding total CREB signals from the same blot. Student's paired t test was used. B, Mixed neuronal cultures were treated as described in A and analyzed by immunocytochemistry for the presence of Ser133-phosphorylated CREB in the nucleus (green). The neuronal cell population was identified with a MAP2-specific antibody (red). Nuclei were visualized using DAPI (blue). In the absence of Reelin and glutamate, two of a total of 69 scored nuclei from MAP2-expressing neurons stained positive for phospho-CREB (2 of 69), in the presence of Reelin alone (7 of 67), in the presence of glutamate alone (9 of 64), and in the presence of Reelin and glutamate (59 of 78). The number of phospho-CREB-positive nuclei after glutamate exposure was thus significantly increased in the presence of Reelin (p < 0.000001).

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