Dcx reexpression reduces subcortical band heterotopia and seizure threshold in an animal model of neuronal migration disorder

Abstract

Disorders of neuronal migration can lead to malformations of the cerebral neocortex that greatly increase the risk of seizures. It remains untested whether malformations caused by disorders in neuronal migration can be reduced by reactivating cellular migration and whether such repair can decrease seizure risk. Here we show, in a rat model of subcortical band heterotopia (SBH) generated by in utero RNA interference of the Dcx gene, that aberrantly positioned neurons can be stimulated to migrate by reexpressing Dcx after birth. Restarting migration in this way both reduces neocortical malformations and restores neuronal patterning. We further find that the capacity to reduce SBH continues into early postnatal development. Moreover, intervention after birth reduces the convulsant-induced seizure threshold to a level similar to that in malformation-free controls. These results suggest that disorders of neuronal migration may be eventually treatable by reengaging developmental programs both to reduce the size of cortical malformations and to reduce seizure risk.

Figure 1. Temporal control of Dcx expression in mis-positioned neurons by using Cre-dependent expression vectors and 4-OHT-activatable Cre recombinase

(a) A schematic of the experimental approach used to test the hypothesis that re-expression of Dcx can reactivate migration and regress malformation. (b) A 4-OHT-activatable Cre recombinase composed of two ER binding domains is expressed under the control of the CAG promoter (CAG-ER^T2CreER^T2). Cre-dependent Dcx expression vector contains neo with a stop codon flanked by loxP sites (CALNL-DCX-eGFP). In the presence of 4-OHT, recombination occurs and DCX-eGFP is expressed. (c) Animals were electroporated at E14 with 4 plasmids: CAG-mRFP, CAG-ER^T2CreER^T2, CALNL-DCX-eGFP and 3UTRhp. In 4-OHT-treated animals (left panels), DCX-GFP is expressed and Dcx is detected with antibodies in transfected mis-positioned neurons. In vehicle-treated animals (right panels), no signal is detected in the green channel or with Dcx antibodies. (d) Transfection conditions. (e) Scheme of experiments. Scale bars, 50µm.

Figure 2. Restoration of neocortical lamination and regression of SBH after re-expression of Dcx at P0

(a) Representative frontal neocortical sections showing laminar position of transfected cells in animals electroporated at E14 with either effective (3UTRhp) (bottom panels) or non-effective (3UTRm3hp) (top panels) DCX-targeting shRNA vectors together with CAG-mRFP, and sacrificed postnatally from P0 to P15. (b) Representative frontal neocortical section showing restoration of neocortical lamination at P20 after re-expression of Dcx in mis-positioned neurons at P0. 4 plasmids were electroporated at E14: CAG-mRFP, CAG-ER^T2CreER^T2, CALNL-DCX-eGFP and 3UTRhp, and 4-OHT was administered at birth. (c) Quantification of transfected cells distribution within the neocortical grey matter after induction of eGFP or DCX-eGFP expression in animals electroporated at E14 with either effective (3UTRhp) or non-effective (3UTRm3hp) Dcx-targeting shRNA vectors, together with CA-mRFP, CAG-ER^T2CreER^T2, and CALNL-DCX-eGFP or CALNL-eGFP (10–12 sections from 2–3 animals per condition). (d) Size and position of SBH at 3 rostro-caudal levels after induction of eGFP (top) or DCX-eGFP (bottom) expression in animals electroporated at E14 with CAG-mRFP, CAG-ER^T2CreER^T2, 3UTRhp, and CALNL-eGFP (top) or CALNL-DCX-eGFP (bottom). (e) Quantification of SBH surface after induction of eGFP or DCX-eGFP in the same experimental conditions (8–9 sections from 2–3 animals per condition). *** P < 0.001, * P < 0.05. Scale bars, 500 µm (a, b)

Figure 3. Migration of Fated Upper Layer neurons from SBH

(a) A scheme of experiment. Four plasmids were electroporated at E14: CAG-mRFP, CAG-ER^T2CreER^T2, CALNL-DCX-eGFP and 3UTRhp, and 4-OHT or its vehicle solution was administered at birth. DCX-eGFP expression is induced in the 4-OHT-treated group only. (b) Immunohistochemistry of P20 neocortical sections showing E14 transfected and non-transfected cells immunopositive for the upper layers neurons marker CDP/Cux1 (d, h) within SBH at P20, in the absence of Dcx re-expression (left panels), or after Dcx re-expression (right panels). (c–d) Quantifications of surface (c) and density (d) of CDP/Cux1+ cells within SBH in the same experimental conditions (6 sections from 2 animals per condition). *** P < 0.001, ** P< 0.01. Scale bar, 150 µm.

Figure 4. Morphology of rescued neurons

(a, b) Immunohistochemistry for the upper layers neurons marker CDP/Cux1 on P20 frontal neocortical sections. Animals were electroporated at E14 with either non-effective (3UTRm3hp) (a) or effective (3UTRhp) (b) Dcx-targeting shRNA vectors together with CAG-mRFP, CAG-ER^T2CreER^T2, and CALNL-eGFP (a) or CALNL-DCX-eGFP (b), and injected with 4-OHT at birth. Both initially correctly positioned transfected neurons (a) and initially mis-positioned transfected neurons induced to migrate to appropriated positions after Dcx re-expression (b) (both green and red) are located within the CDP/Cux1+ band of upper layers neurons (in blue). (c, d) Reconstructed cortical neurons showing dendritic arborization in the same experimental conditions. (e–g) Quantifications of the dendritic arborization: mean number of apical processes per neurons (e), their mean length (f) and total length (g) (36–37 reconstructed neurons from 3–4 animals per condition). Scale bars, 200 µm (a).

Figure 5. Critical developmental period for regressing malformation

(a–e) Representative reconstructed neocortical sections showing the laminar position of neurons at P20 after induction of eGFP (a, b) or DCX-eGFP expression at P0 (a–c), P5 (d) and P10 (e). Black dots represent positions of transfected cells. 4 plasmids were electroporated at E14: CAG-mRFP (a–e), CAG-ER^T2CreER^T2 (a–e), CALNL-eGFP (a, b) or CALNL-DCX-eGFP (c–e), and 3UTRhp (c–e) or 3UTRm3hp (a), and 4-OHT was administered at P0 (a–c), P5 (d) and P10 (e). (f, g) Quantification of transfected cells distribution within the neocortical grey matter after induction of eGFP or DCX-eGFP expression at P5 (f) and P10 (g), compared to Dcx re-expression at P0 (8–12 sections from 2–3 animals per condition). (h) Size and position of SBH at 3 rostro-caudal levels after induction of Dcx expression at P5 (top) and P10 (bottom). (i) Quantification of SBH surface after induction of Dcx expression at P5 and P10 (8 sections from 2–3 animals per condition). *** P < 0.001, ** P < 0.01, * P < 0.05. Scale bar, 500 µm.

Figure 6. Decreased Seizure Susceptibility to PTZ-induced seizures

Quantification of doses of PTZ (a), latency to induce generalized tonic-clonic seizures (b) and interval between minimal and generalized seizures (c) in 3 groups of animals: Malformation free animals (ineffective shRNAs and induced eGFP expression induced at P0), double cortex animals with SBH and lamination deficits (effective shRNAs and eGFP expression induced at P0) and P0 rescue animals with restored lamination and regressed SBH (effective shRNAs and DCX-eGFP expression induced at P0). (d) Example of a double cortex animal experiencing rearings, fallings and convulsions (top panels) and a P0 rescue animal at the same dose of PTZ and same time after injection (bottom panels). The P0 rescue animal experienced seizures 10 minutes later, after an additional injection of PTZ was given. *** P < 0.001, ** P < 0.01, * P < 0.05.