Reelin and cyclin-dependent kinase 5-dependent signals cooperate in regulating neuronal migration and synaptic transmission

Abstract

Neuronal migration and positioning in the developing brain require the coordinated interaction of multiple cellular signaling pathways. The extracellular signaling molecule Reelin and the cytoplasmic serine/threonine kinase Cdk5 (cyclin-dependent kinase 5) are both required for normal neuronal positioning, lamination of the neocortex, and foliation of the cerebellum. They also modulate synaptic transmission in the adult brain. It is not known, however, to what extent Cdk5 participates in Reelin signaling and whether both pathways interact on the genetic or biochemical level. We have used genetically altered mice to generate compound functional defects of Reelin and Cdk5 signaling. Differential neurohistochemical staging combined with the biochemical analysis of Reelin- and Cdk5-dependent signaling in primary embryonic neurons and electrophysiology in hippocampal slices reveals evidence for genetic and functional interaction between both pathways. Inhibition of Reelin or Cdk5 signaling had no discernible biochemical effect on each other. Taken together, these findings suggest that both pathways function together in a parallel, rather than a simple, linear manner to coordinate neuronal migration and neurotransmission in the developing and mature brain.

Figure 1.

Synergistic genetic interaction of p35 and apoER2 on neuronal positioning in the hippocampus. Sagittal sections of the hippocampal region of 21-d-old adult mice were stained with hematoxylin and eosin. Representative sections of wild-type (A), reelin-/- (B), Apoer2-/- (C), Vldlr-/- (D), p35-/- (E), p39-/- (F), p35-/-;Apoer2-/- (G), p39-/-;Apoer2-/- (H), p35-/-;Vldlr-/- (I), and p39-/-;Vldlr-/- (J) mutant mice are shown. The wild-type hippocampus (A) at P21 shows distinct neuronal layering with a tightly packed pyramidal cell layer of the CA region as well as the dentate gyrus. Both reelin-/- (B) and double Apoer2-/-;Vldlr-/- (data not shown) hippocampus display severely disrupted cell layering with a very diffuse CA cell layer and a poorly defined dentate gyrus. Mice deficient in Apoer2 (C) and p35 (E) display disorganization of the CA field with loosely packed cells as well as noticeable cells rifts. The hippocampal formation of Vldlr-/- (D), p39-/- (F), and p39-/-;Vldlr-/- (J) mice are comparable with that of wild-type mice. The hippocampus of p35-/-;Apoer2-/- double mutants (G) are more severely affected than either corresponding single mutant, displaying a much more diffuse packing of cells of the CA region more closely resembling that of the Reelin-deficient mouse (B). The dentate gyrus is also less organized. The phenotype of the p39-/-;Apoer2-/- (H) hippocampus seems comparable with that of the single Apoer2-/- mutant (C), whereas the appearance of the p35-/-;Vldlr-/- (I) hippocampus closely resembles that of the single p35-/- mutant (E). Six samples per genotype were examined, and one representative sample is shown.

Figure 2.

Neuronal positioning in the cerebral cortex is disrupted in p35;Vldlr and p35; Apoer2 double mutant mice. Sagittal sections of 21-d-old adult mice were stained with hematoxylin and eosin. A, In wild-type cortex, a distinct laminar organization is apparent, featuring a distinct layer I, followed by neuronal layers II through VI. B, Vldlr-deficient mice do not show any apparent abnormality in cortical lamination, whereas mild disruptions are visible in the Apoer2-deficient (C) and p35-deficient (D) cortex. The p35;Vldlr (E) and p35;Apoer2 (F) double knock-out mice exhibit noticeable disruptions in cortical neuronal positioning, most apparent by the infiltration of cells into layer I. One representative sample is shown from a total of six samples analyzed.

Figure 3.

Genetic deficiency of p35 and Apoer2 does not prevent Reelin-induced Dab1 and PKB phosphorylation. Mouse embryonic neurons were prepared from E16 embryos derived from the mating of p35+/-; Apoer2+/- and p35+/-; Apoer2-/- mice, generating the genotypes shown at the bottom. After 4 d in culture, stimulation with exogenously applied Reelin for 20 min (lanes 2, 4, 6, and 8) increased tyrosine phosphorylation of Dab1 (p80, as detected by 4G10 antibody) and serine phosphorylation of PKB at Ser473 in neurons irrespective of genotype. Total levels of Dab1 and PKB as well as Cdk5 remained unchanged across the different genotypes and treatment with Reelin. Protein levels of apoER2 and p35 confirm results obtained from genotype analysis (bottom).

Figure 4.

Pharmacological or genetic inhibition of Cdk5 activity does not affect Reelin-induced Dab1 and PKB phosphorylation. A, Mouse embryonic neurons from E16 embryos were incubated with the Cdk5 inhibitors olomoucine and roscovitine 1 hr before Reelin treatment at the concentrations indicated at the top. Reelin-induced Dab1, PKB, and GSK-3β phosphorylation appeared unaffected at concentrations ranging from 10 to 100 μm olomoucine (lanes 3-8) and from 10 to 100 μm roscovitine (lanes 9-14). Total protein levels of Cdk5 and apoER2 remained unchanged across all tested conditions. B, Reelin-induced signaling is not inhibited in the absence of Cdk5 activity. Mouse embryonic neurons were derived from E16 embryos from the mating of p35-/-;p39+/- and p35+/-; p39-/- mice, generating the genotypes shown at the bottom.

Figure 5.

Cdk5 substrates are not affected by Reelin signaling. Primary cortical neurons from p35;p39 compound mutant mice (bottom) were stimulated with control and Reelin conditioned medium. Cell lysates were then blotted for phospho-serine and phospho-threonine cyclin-dependent kinase (CDK) substrates. Arrows indicate Cdk5-specific substrates.

Figure 6.

Electrophysiological responses at Schaffer collateral synapses in area CA1 of hippocampus. A, Short-term plasticity assessed by PPF at 25°C demonstrated a clear defect in mice deficient in p39 protein (○) compared with wild-type mice (•). Results are shown as mean ± SEM B, LTP induction is reduced modestly in p39 mutant mice. LTP induced with two trains of a 1 sec 100 Hz stimulation separated by 20 sec (arrow) is shown for mice deficient in p39 (○) or p35 (□) and compared with that of wild-type mice(•). LTP induction is mildly impaired in p39 knock-out mice and unchanged in p35 knock-outs relative to LTP from wild-type control mice. Results are shown as mean ± SEM. In this and the following figures, data are normalized to the average of the initial 20 min baseline value (defined as 100%).

Figure 7.

A potential mechanism by which Reelin signaling and Cdk5 might act synergistically in coordinating neuronal migration. Reelin signaling takes place at the neuronal growth cone (Beffert et al., 2002), which is also the first part of the migrating neuron to make contact with Reelin at the boundary to the marginal zone. Tyrosine-phosphorylated Dab1 is transported retrogradely along the microtubule tracks and contacts Lis1 in the perinuclear region and in the microtubule organizing center. Synergistic interaction of tyrosine-phosphorylated Dab1 with Lis1, as well as Cdk5-dependent phosphorylation of Nudel, and potentially other components of the nuclear distribution machinery such as microtubule-associated proteins like tau and mitogen-activated protein kinases, may be required to fully activate the molecular motors that drive the translocation of the nucleus to the superficial layers of the developing cortex. We, thus, propose that the reduced signal input at the level of Dab1 phosphorylation, combined with a reduction in Cdk5 activity, might effectively prevent a functionally meaningful activation of the required motor complex. This would explain the apparent complete inability of neurons with partial defects in Reelin and Cdk5 pathways to translocate to their proper positions in the cortex, generating a close phenocopy of reeler in p35;Apoer2 double deficient hippocampus and cortex.