Involvement of Calcium-Dependent Pathway and β Subunit-Interaction in Neuronal Migration and Callosal Projection Deficits Caused by the Cav1.2 I1166T Mutation in Developing Mouse Neocortex

Abstract

Introduction: Gain-of-function mutations in the L-type Ca²⁺ channel Cav1.2 cause Timothy syndrome (TS), a multisystem disorder associated with neurologic symptoms, including autism spectrum disorder (ASD), seizures, and intellectual disability. Cav1.2 plays key roles in neural development, and its mutation can affect brain development and connectivity through Ca²⁺-dependent and -independent mechanisms. Recently, a gain-of-function mutation, I1166T, in Cav1.2 was identified in patients with TS-like disorder. Its channel properties have been analyzed in vitro but in vivo effects of this mutation on brain development remain unexplored. Methods: In utero electroporation was performed on ICR mice at embryonic day 15 to express GFP, wild-type, and mutant Cav1.2 channels into cortical layer 2/3 excitatory neurons in the primary somatosensory area. The brain was fixed at postnatal days 14-16, sliced, and scanned using confocal microscopy. Neuronal migration of electroporated neurons was examined in the cortex of the electroporated hemisphere, and callosal projection was examined in the white matter and contralateral hemisphere. Results: Expression of the I1166T mutant in layer 2/3 neurons caused migration deficits in approximately 20% of electroporated neurons and almost completely diminished axonal arborization in the contralateral hemisphere. Axonal projection in the white matter was not affected. We introduced second mutations onto Cav1.2 I1166T; L745P mutation blocks Ca²⁺ influx through Cav1.2 channels and inhibits the Ca²⁺-dependent pathway, and the W440A mutation blocks the interaction of the Cav1.2 α1 subunit to the β subunit. Both second mutations recovered migration and projection. Conclusion: This study demonstrated that the Cav1.2 I1166T mutation could affect two critical steps during cerebrocortical development, migration and axonal projection, in the mouse brain. This is mediated through Ca²⁺-dependent pathway downstream of Cav1.2 and β subunit-interaction.

Keywords: Timothy syndrome; calcium channel; callosal projection; neocortex; radial migration.

FIGURE 1

Diagram of signal pathways downstream of Cav1.2 that regulate gene expression. The I1166T and four additional mutations used in this study are also shown.

FIGURE 2

Expression of the Cav1.2 I1166T mutant caused neuronal migration deficit. (A) Left, GFP images of electroporated cortex in the GFP-alone group. Cyan lines represent the top; pial surface, the bottom edge of the GFP-positive neuron cluster, and the top and bottom edge of white matter (see section “Materials and Methods”). Right, histogram of radial index (RI) of the GFP-positive neurons. Colors, each sample. Black, mean ± SEM. Y-axis is enlarged and shown at the bottom. (B,C) Images and histograms of the Cav1.2^WT group (B) and Cav1.2^I1166T group (C), as shown in (A). (D) Histogram of the mean ± SEM are merged for GFP-alone, Cav1.2^WT, and Cav1.2^I1166T groups. (E) Proportion of correctly migrated neurons (top) and migration-failed neurons in WM and Middle (bottom). Independent values and mean ± SEM are shown. ***P < 0.001; *P < 0.05; n.s., P ≥ 0.05.

FIGURE 3

Blockade of Ca²⁺ influx and β subunit-interaction improves migration deficit caused by the I1166T mutation. (A,B) GFP images and histograms of the Cav1.2^I1166T/L745P (A) and Cav1.2^I1166T/W440A group (B), as shown in Figure 2A. (C) Histogram of the mean ± SEM are merged for Cav1.2^WT, Cav1.2^I1166T/W440A, Cav1.2^I1166T/L745P, and Cav1.2^I1166T groups. (D) Proportion of correctly migrated neurons (left) and that of migration-failed neurons located in WM and Middle (right). Independent values and mean ± SEM are shown. ***P < 0.001; **P < 0.01; n.s., P ≥ 0.05.

FIGURE 4

Blockade of CaM-binding but not CaN-binding rescues the migration deficit. (A,B) GFP images and histograms of the Cav1.2^{I1166T/I1624A} (A) and Cav1.2^{I1166T/A1929P} group (B), as shown in Figure 2A. (C) Histogram of the mean ± SEM are merged for Cav1.2^WT, Cav1.2^{I1166T/I1624A}, Cav1.2^{I1166T/A1929P}, and Cav1.2^I1166T/L745P groups. (D) Proportion of correctly migrated neurons (left) and that of migration-failed neurons located in WM and Middle (right). Independent values and mean ± SEM are shown. **P < 0.01; *P < 0.05; n.s., P ≥ 0.05.

FIGURE 5

Axonal arborization in the contralateral cortex is affected by I1166T mutation, which is rescued by the blockade of calcium overload and β subunit-interaction. (A) Axonal arborization of the GFP-alone group. (A1) An example image of axonal arborization in the contralateral somatosensory area. (A2) Binarized images around the S1/S2 border of multiple samples were averaged and are shown. (A3) Proportion of pixels that have axonal signals are shown against the radial position. Colors for each sample: black, the mean ± SEM. (A4) Maximum proportion in the white matter (WM; Radial index = 0–0.1), layer 5 (L5; Radial index = 0.4–0.6), and upper layers (L1–3; Radial index = 0.8–1) of each sample; the mean ± SEM are shown. (B,C) Axonal arborization of Cav1.2^WT- (B) and Cav1.2^I1166T-expressing neurons (C) are shown as in (A). (D) Proportions that were normalized with the maximum proportion in the WM are shown for the GFP-alone, Cav1.2^WT, and Cav1.2^I1166T groups by the mean ± SEM. (E) Proportions for Cav1.2^I1166T/L745P, Cav1.2^I1166T/W440A, Cav1.2^{I1166T/I1624A}, and Cav1.2^{I1166T/A1929P} groups are shown as in (D). (F) Proportion in L5 (left) and L1–3 (right) for GFP, WT, and all mutation groups used in this study are shown by the mean ± SEM. ***P < 0.001; *P < 0.05; n.s., P ≥ 0.05.

FIGURE 6

Local axonal arborization in the ipsilateral cortex is not affected by the expression of Cav1.2 mutants. (A) Left, An example GFP image in the electroporated hemisphere of GFP-alone group. A strong GFP signal was seen in the upper layer, representing cell bodies, dendrites, and axons of GFP-expressing neurons. A strong signal was also seen in the middle of the cortex, representing axons of neurons migrated to the L2/3. Right, the medians of GFP intensities are calculated along the tangential axis of the images. Colors, each sample. Black, the mean ± SEM. (B,C) GFP images and median intensities of Cav1.2^WT group (B) and Cav1.2^I1166T group (C), as shown in (A). Because migration-failed neurons were sparse in the middle of the cortex, their strong signals could be excluded and axonal signals could be obtained by calculating the medians. (D) The values of mean ± SEM of three groups are merged.

FIGURE 7

Axonal projection in the white matter is not affected by the Cav1.2 I1166T mutation. (A) Left, GFP images of white matter in the GFP-alone group. Yellow squares represent the four areas shown on the right. Right, GFP images of Ipsi, Midline, Contra, and Contra2 areas. Intensities of each area were averaged in the horizontal axis and shown on the right. Gaussian fit to the intensity distribution is shown in red. (B,C) Axonal projections of the Cav1.2^WT (B) and Cav1.2^I1166T (C) groups are shown as in (A). (D) GFP intensities normalized by that of Ipsi are shown for each sample and the mean ± SEM for the GFP-alone, Cav1.2^WT, and Cav1.2^I1166T groups. All the combination of groups had insignificant differences (P > 0.09). (E) Broadness of axon bundles in white matter, represented by the standard deviation of Gaussian curves, are shown for each sample and the mean ± SEM for the GFP-alone, Cav1.2^WT, and Cav1.2^I1166T groups. All the combination of groups had insignificant differences (P > 0.1).