Deletion of ERK2 mitogen-activated protein kinase identifies its key roles in cortical neurogenesis and cognitive function - PubMed (original) (raw)

Deletion of ERK2 mitogen-activated protein kinase identifies its key roles in cortical neurogenesis and cognitive function

Ivy S Samuels et al. J Neurosci. 2008.

Abstract

The mitogen-activated protein (MAP) kinases ERK1 and ERK2 are critical intracellular signaling intermediates; however, little is known about their isoform-specific functions in vivo. We have examined the role of ERK2 in neural development by conditional inactivation of the murine mapk1/ERK2 gene in neural progenitor cells of the developing cortex. ERK MAP kinase (MAPK) activity in neural progenitor cells is required for neuronal cell fate determination. Loss of ERK2 resulted in a reduction in cortical thickness attributable to impaired proliferation of neural progenitors during the neurogenic period and the generation of fewer neurons. Mutant neural progenitor cells remained in an undifferentiated state until gliogenic stimuli induced their differentiation, resulting in the generation of more astrocytes. The mutant mice displayed profound deficits in associative learning. Importantly, we have identified patients with a 1 Mb microdeletion on chromosome 22q11.2 encompassing the MAPK1/ERK2 gene. These children, who have reduced ERK2 levels, exhibit microcephaly, impaired cognition, and developmental delay. These findings demonstrate an important role for ERK2 in cellular proliferation and differentiation during neural development as well as in cognition and memory formation.

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Figures

Figure 1.

Figure 1.

Generation of ERK2 conditional knock-out mice. A, Schematic representation of gene targeting strategy. The shaded boxes on the _mapk1/ERK2 locus and the targeting construct designate homologous regions identified for recombination. Numbered boxes represent mapk1 exons 2–4. The targeting construct contains a floxed Neo cassette upstream of exon 2 and a loxP site between exons 2 and 3. A βADT cassette was inserted downstream of exon 3 for negative selection. Relevant restriction sites and the 3′ probe used for Southern blot analysis are indicated. Recombinant ES cells were transfected with a Cre plasmid to remove the Neo cassette, giving rise to the floxed mapk1 allele. Cre recombinase expression from the h_GFAP–cre transgene produces a null allele. B, Southern blot of genomic brain DNA. After Xba digestion of DNA, the 3′ probe identifies the wild-type allele as a 4.7 kb fragment and the mutant allele as 11.5 kb before and 9.5 kb after recombination. C, Western blot analysis of adult cortical brain homogenates. Tissue was homogenized, separated by SDS-PAGE, and immunoblotted with anti-ERK1, anti-ERK2, and β-tubulin antibodies. D, Western blot analysis of embryonic cortical homogenates. Tissue was lysed, separated by SDS-PAGE, and immunoblotted with an antibody directed to an epitope at the N terminus of ERK2. A lack of bands below 42 kDa in both flox/flox and flox/flox cre+ samples demonstrates that no truncated protein fragments were produced by recombination. An antibody for c-Src was used as a loading control.

Figure 2.

Figure 2.

ERK2 CKO mice display reduced cortical thickness. A, Dorsal view of P2 CKO (bottom) and wild-type (top) littermates. B, Quantitative analysis of cortical thickness. Corresponding coronal sections from CKO and wild-type brains were used to measure thickness of the frontal, parietal, temporal, and occipital lobes corresponding to the motor, somatosensory, auditory, and visual cortices. Measurements were taken from the ventricular zone to the pial surface (n = 5). Two-way ANOVA, p < 0.0001 with Bonferroni's post hoc tests, **p < 0.01, ***p < 0.001. C, Representative coronal sections of CKO and wild-type brains from P2, stained with DAPI. Note the difference in cellular density apparent between genotypes. D, Coronal sections of adult ERK1−/− and wild-type brains stained with the fluorescent Nissl stain Neurotrace.

Figure 3.

Figure 3.

Loss of ERK2 expression and activity in ERK2 CKO cortices. A–D, E14.5 and E16.5 cortices were microdissected and prepared for Western blot analysis immediately during dissection. The levels of ERK2, ERK1, and phospho-ERK1/2 were detected by immunoblotting, and representative blots are shown. Densitometric analysis of the bands, normalized to β-tubulin, was performed for quantification and expressed as fold difference relative to wild-type. For A, quantification of ERK2 levels at E14.5 (CKO, n = 15; WT, n = 21; p < 0.0001) and E16.5 (CKO, n = 7; WT, n = 8; p < 0.0001). B, Quantification of ERK1 expression levels at E14.5 (CKO, n = 13; WT, n = 18; p = 0.4019) and E16.5 (CKO, n = 7; WT, n = 8; p = 0.7772). C, Relative phospho-ERK2 activation at E14.5 (CKO, n = 12; WT, n = 18; p = 0.0093) and E16.5 (CKO, n = 7; WT, n = 8; p < 0.0001). D, Relative phospho-ERK1 activation at E14.5 (CKO, n = 11; WT, n = 17; p = 0.3594) and E16.5 (CKO, n = 4; WT, n = 4; p < 0.0001). **p < 0.001; ***p < 0.0001.

Figure 4.

Figure 4.

Inactivation of ERK2 in neural progenitor cells results in the generation of fewer cortical neurons. A, Analysis of cell density and cell fate in CKO and wild-type littermates at P10. Corresponding coronal sections of P10 cortices were immunostained with NeuN (green) and counterstained with DAPI (blue). Total cell number per cortical region from the VZ to the pial surface was calculated by counting DAPI+ cells in both medial (primary motor cortex, CKO, 1386.167 average cells per region; WT, 1398.167 average cells per region; for each n = 3) and lateral (primary somatosensory cortex, CKO, 1534.833 average cells per region; WT, 1571.167 average cells per region; for each n = 3) sections. Two-way ANOVA, p = 0.6179, and Bonferroni's post hoc test, p > 0.05. The fold difference in NeuN+ cells was calculated by two-way ANOVA, p < 0.0001, Bonferroni's post hoc test, p < 0.0001. Fold difference in DAPI; NeuN− cells was calculated. Two-way ANOVA, p = 0.0004, Bonferroni's post hoc test, p < 0.01. B–D, Corresponding coronal sections of P2 CKO (left) and WT (right) littermates were stained with anti-Tbr1 (B, green), anti-Otx1 (C, red), and anti-Brn1 (D, red) antibodies and counterstained with DAPI. Cortical lamina were identified based on morphology and distance from the pial surface. Immunoreactive cells were counted from two independent reference spaces of the cortex from at least eight sections per genotype. For each, two-way ANOVA, p < 0.0001, and Bonferroni's post hoc tests, p < 0.0001, were performed (n = 4). **p < 0.001; ***p < 0.0001.

Figure 5.

Figure 5.

Inactivation of ERK2 in neural progenitor cells results in the presence of more astrocytes within the cerebral cortex. A, B, Representative images of corresponding coronal sections from neonatal (A) and mature (B) ERK2 CKO and wild-type littermates immunostained with anti-Zebrin II (red) anti-GFAP (green) antibodies and counterstained with DAPI. A higher-magnification image is shown on the right. C, Primary astrocyte cultures from CKO and wild-type P2 pups were prepared and maintained for 5 DIV. Cultures were incubated with BrdU overnight and fixed. Quantification of percentage BrdU immunoreactivity (CKO, n = 4; WT, n = 3; p = 0.9084). D, Quantification of Ki67-immunoreactive cells (CKO, n = 3; WT, n = 3; p = 0.4166). E, Characterization of astrocyte proliferative index (CKO, n = 2; WT, n = 2; p = 0.9827). F, MTT viability assay (CKO, n = 4; WT, n = 5; p = 0.4556).

Figure 6.

Figure 6.

ERK2 CKO mice display changes in the dynamics of NPC proliferation. A–C, Pregnant mice were given intraperitoneal injections of BrdU at E12.5, E14.5, and E16.5, respectively. Embryos were fixed 30 min after BrdU injection and corresponding coronal sections of ERK2 CKO and wild-type littermates were double labeled with anti-pH3+ (red) and anti-BrdU (green) antibodies followed by DAPI counterstaining (blue). Confocal images were taken at 10× and 20× (A, B; scale bar, 50 μm) and 10× and 40× (C; scale bar, 20 μm). D, E, Corresponding coronal sections of E14.5 (D) and E16.5 (E) embryos were immunostained with Tbr2 and phosphorylated histone H3 followed by counterstaining with DAPI. Scale bar, 20 μm.

Figure 7.

Figure 7.

ERK2 CKO cortical progenitor cells exhibit reduced neuronal generation. A, Cortical progenitor cell cultures from ERK2 CKO and wild-type E14.5 embryos were grown in Neurobasal with FGF2 for 2 d in vitro. Cells were then fixed and immunostained. Progenitors were identified by Nestin immunoreactivity (A; n = 3). Cultures were assayed for immature neurons with βIII-tubulin (B; n = 3), mature neurons with MAP2 (C; n = 4), and astrocyte precursors with S100β (D; n = 4). E, Analysis of cellular identity in culture with FGF2. Fold difference is the comparison of average of positive cells/total cell number relative to wild-type. Student's t test, p < 0.001 for Nestin; p = 0.014 for βIII-tubulin; p = 0.0003 for MAP2; and p = 0.0018 for S100β. *p < 0.05; **p < 0.001; ***p < 0.0001.

Figure 8.

Figure 8.

ERK2 CKO cortical progenitors generate more astrocytes in the presence of gliogenic stimuli. Cortical progenitor cell cultures from ERK2 CKO and wild-type E14.5 embryos were grown in Neurobasal media. Twelve hours after plating, cells were treated with CNTF to induce astrocyte differentiation. Cells were fixed and immunostained with anti-vimentin (A), anti-GFAP (B), and anti-S100β (C) antibodies after 2 d in vitro (1, 2) and 4 d in vitro (3, 4). D–F, Analysis of cellular identity with CNTF treatment. Fold difference in astrocyte generation was measured by the average of marker immunopositive cells/total cell number relative to wild-type at 2 DIV and 4 DIV (n = 4 for each). Two-way ANOVA, p < 0.0001 with Bonferroni's post hoc tests, p < 0.0001 for each. **p < 0.001; ***p < 0.0001.

Figure 9.

Figure 9.

Male ERK2 CKO mice have deficits in associative learning. Male ERK2 CKO mice displayed deficits in cued and contextual fear conditioning. A, At 24 h after training, CKOs showed significantly less freezing behavior when tested for contextual and cued recall (t(16) = 2.27 and 2.95, respectively; p < 0.05). B, Repeated training, or “overtraining,” did not overcome the deficit seen in cued and contextual fear conditioning. CKOs showed significantly less freezing behavior in both recall tests when tested the next day (t(16) = 3.65 and 3.35, respectively; p < 0.05). *p < 0.05.

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