ERK inhibition rescues defects in fate specification of Nf1-deficient neural progenitors and brain abnormalities

Abstract

Germline mutations in the RAS/ERK signaling pathway underlie several related developmental disorders collectively termed neuro-cardio-facial-cutaneous (NCFC) syndromes. NCFC patients manifest varying degrees of cognitive impairment, but the developmental basis of their brain abnormalities remains largely unknown. Neurofibromatosis type 1 (NF1), an NCFC syndrome, is caused by loss-of-function heterozygous mutations in the NF1 gene, which encodes neurofibromin, a RAS GTPase-activating protein. Here, we show that biallelic Nf1 inactivation promotes Erk-dependent, ectopic Olig2 expression specifically in transit-amplifying progenitors, leading to increased gliogenesis at the expense of neurogenesis in neonatal and adult subventricular zone (SVZ). Nf1-deficient brains exhibit enlarged corpus callosum, a structural defect linked to severe learning deficits in NF1 patients. Strikingly, these NF1-associated developmental defects are rescued by transient treatment with an MEK/ERK inhibitor during neonatal stages. This study reveals a critical role for Nf1 in maintaining postnatal SVZ-derived neurogenesis and identifies a potential therapeutic window for treating NF1-associated brain abnormalities.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Figure 1. Nf1-deficient adult brain exhibits enlarged corpus callosum and reduced olfactory bulb

(A, A’, B) Whole-mount images and size quantification of control and Nf1hGFAPCKO brains and OB. (C, C’) Schematic demonstration of different histological planes used for analysis and the SVZ/RMS/CC/OB system and the orientation of brains analyzed (A, anterior; P, posterior; D, dorsal; V, ventral). (D) Sagittal sections from control and Nf1hGFAPCKO brains at 2 months were analyzed at P2 position, stained with hematoxylin & eosin (H&E) and imaged at 3 increasingly higher magnifications. The cells that form chain-like structures (arrowheads) were exclusively observed in the mutant CC. (E) The thickness of the control and mutant CC was quantified. (F, G) Coronal sections from control and mutant brains were stained with NF200/Olig2 and MBP/Olig2. Lower panels in (F) are the high magnification view of delineated CC areas in the upper panels. (H, I) Ultrathin sections were subjected to EM analysis from dorsal to ventral CC. Representative EM images from two histological planes are shown in (H) and (I). * indicates degenerating tissues only observed in the mutant CC. (J, K) Two sets of EM images covering the entire thickness of the CC at two parallel positions (column 1 and column 2) at comparable histological planes were analyzed. The total number of axons (J) and cell bodies (K) in each column from 3 controls and 3 mutants were quantified. (L) Total cell density in the CC was quantified and compared at P1, P2, P3 and M positions based on H&E-stained sections. (M) Control and mutant CC were stained for GFAP/Olig2, and their density was quantified in (N). The number of cells expressing both GFAP and Olig2 (arrows, M) was significantly increased in mutant CC (O). Dashed lines delineate the border of the CC. All the quantification data are presented as mean ± SEM. LV, *, lateral ventricle. Scale bars: 50 μm except for (H) and (I) at 2 μm. See also Figure S1.

Figure 2. Nf1hGFAPCKO SVZ shows increased glial differentiation and ectopic Olig2 expression in Ascl1+ TAP/IPCs

(A) Representative sections of the control and mutant SVZ at P2 position were stained with H&E and imaged at 3 magnifications (a-b”). The high magnification views of the boxed area in (a) and (b) are shown in (a’, a’’) and (b’, b’’), respectively. Adjacent sections were stained for Nestin/GFAP (c, d), Olig2/NG2 (e, f) and GFAP/Dcx (g, h). Arrows identify Olig2+/NG2+ cells and arrowheads mark Olig2+/NG2- cells (e-f’). (B) The number of cells per unit length of LV at P1-P3 was quantified. (C) The percentage of SVZ cells expressing different lineage markers (Ac-h’) was quantified. (D, E) Olig2/Dlx2/Ascl1 triple immunofluorescence labeling was performed on sections from control and mutant brains. In the ventral (D) and dorsal (E) SVZa, Olig2+/Dlx2+/Ascl1+ triple-positive cells (white) and Olig2+/Dlx2-/Ascl1+ double-positive cells (pink) are marked by arrows and arrowheads, respectively. (F) The percentage of Ascl1+ cells expressing Olig2 and/or Dlx2 was quantified and compared between the control and mutant SVZ. All the quantification data are presented as mean ± SEM. LV and *, lateral ventricle. St, striatum. Scale bars: 50 μm. See also Figures S2 and S3.

Figure 3. Nf1 inactivation leads to increased gliogenesis in the CC at the expense of neurogenesis in the OB

A BrdU pulse-chase assay from P60-P90 was performed to label newly differentiated neurons and glia. (A) Low magnification view and (B) the quantification of the overall distribution of BrdU+ cells in P90 control and mutant brains. Arrows identify BrdU+ cells in the CC and arrowheads mark the abnormal increase of BrdU+ cells in the mutant RMS. The dashed line marks the border of the OB and the forebrain. (C) Increased Olig2+/BrdU+ cells (arrows) were observed in the mutant CC compared to controls. (D, D’) A short-term BrdU proliferation assay was performed on P60 mice. (E) 30 days after BrdU pulse, the identity of BrdU+ cells in the RMS was revealed by triple or double labeling of BrdU with Pax6/GFAP (a, b), Dcx/GFAP (c, d), or Olig2 (e, f). Arrows and arrowheads: BrdU double- and single-labeling cells. (F, G) The number and ratio of BrdU+ cells that coexpressed Pax6, GFAP or Olig2 in the control and mutant RMS was quantified. (H) Sections of the control and mutant OB were stained for BrdU, NeuN and DAPI. The number of BrdU+/NeuN+ (arrows) and BrdU+/NeuN- cells per OB surface area was quantified in (H’). All the quantification data are presented as mean ± SEM. *, lateral ventricle. Scale bars: 50 μm. See also Figure S4.

Figure 4. Acute inactivation of Nf1 induces a glia/neuron fate switch in the adult SVZ

(A) The summary of the genotypes of inducible mouse models and the time frame of tamoxifen (TM) induction and analysis. (B) Young adult mice were induced by TM from P26-P30 and analyzed at P31. The cells undergoing Cre-mediated recombination revealed by X-gal staining were restrictedly distributed in the SVZ and RMS. (C-N) Control and mutant brains induced by TM from P26-P30 were analyzed at P60. (C) X-gal staining of the SVZ/RMS/CC system shows the cluster of β-gal+ cells (dashed lines) exclusively identified in the mutant CC. Arrowheads identify individual β-gal+ cells in the CC. (D) The number of β-gal+ cells in the control and mutant SVZ/RMS/CC was quantified. (E, F) The proliferation rate of β-gal+ cells marked by BrdU staining was not significantly different between the control and mutant SVZ. (G-L) The β-gal+ cells coexpressing Olig2 in the control and mutant SVZ (G, H), CC (I, J) and RMS (K, L) are identified by arrows and quantified. Arrowheads label non-colocalizing cells. The inset in (K) shows the coexpression of β-gal and Dcx in the control RMS. Dashed lines delineate the SVZ and/or RMS region. (M) The OB from TM-induced control and Nf1NcreERCKO brains was stained with X-gal and compared. The number of β-gal+ cells was quantified (N). (O) An illustration summarizes the cell lineages targeted by Nestin-creER and their respective derivatives. All the quantification data are presented as mean ± SEM. LV, lateral ventricle. Scale bars: 50 μm. See also Figure S5.

Figure 5. Nf1 inactivation leads to ectopic expression of Olig2 in neural stem/progenitor cells in neonatal brains

(A) Sections from P0.5 control and Nf1hGFAPCKO brains were stained with H&E (a, a’) and Olig2 (b, b’). (B) The percentage of Olig2+ cells in the anterior SVZ (SVZa) was quantified. (C, D) The proliferation rate of Olig2+ cells was not significantly different between the control and mutant SVZa. Arrows and arrowheads: Olig2+/Ki67+ and Olig2+/Ki67-cells. (E, F) The percentage of Ascl1+ cells coexpressing Olig2 and/or Dlx2 in the control and mutant SVZa was analyzed and quantified. Arrows and arrowheads: Olig2+/Dlx2+/Ascl1+ triple-positive cells and Olig2+/Dlx2-/Ascl1+ double-positive cells. (G, H) The expression of Olig2 and Dlx2 in the posterior control and mutant VZ/SVZ and IZ was analyzed and quantified. Dashed lines demarcate the SVZa (A, C, E) or VZ/SVZ regions from the IZ (G). All the quantification data are presented as mean ± SEM. LV and *, lateral ventricle. IZ, intermediate zone. Scale bars: 50 μm. See also Figure S6.

Figure 6. Reduced neurogenesis is accompanied by increased gliogenesis in Nf1hGFAPCKO brains during neonatal SVZ development

(A-C) The size of P0.5 (A) and P8 (B) control and mutant OB was measured and quantified from the dorsal view and the lateral view. (D) The size of P18 control and Nf1hGFAPCKO OB was quantified. (E-K) Control and Nf1hGFAPCKO mice were pulsed with BrdU at P8 and analyzed at P18. (E, F) SVZ-derived neurogenesis was analyzed and quantified by BrdU/NeuN double labeling (arrows) in the granule cell layer (GCL) and periglomerular layer (PGL) of the OB. (G) The density of NeuN+ cells in the OB was quantified. (H-K) The number of newly generated astrocytes (BrdU+/GFAP+) and oligodendrocytes (BrdU+/Olig2+) in the control and mutant CC are illustrated (H, I, arrows) and quantified (J). Arrowheads label non-colocalizing cells. (K) Quantification of the proportion of GFAP+ versus Olig2+ cells in the total BrdU+ cell population in the control and mutant CC. (L, M) Proliferating GFAP+ cells are shown (arrows) and quantified in the control and mutant SVZa at P8. (N, O) The quantification of total cells and the percentage of Ki67+ proliferating cells are shown for P8 control and mutant SVZ. (P, Q) proliferating Olig2+ (Olig2+/Ki67+, arrows) and differentiated Olig2+/Ki67- (arrowheads) cells were shown and quantified in control and mutant P8 SVZa. Dashed lines demarcate the SVZ/RMS. (R) Quantification and characterization of Ascl1+ cells in P8 SVZa based on their expression of Olig2 and/or Dlx2. All the quantification data are presented as mean ± SEM. *, lateral ventricle. Scale bars: 1 mm (A, B); 50 μm (E-P). See also Figure S7.

Figure 7. Neonatal MEKi treatment rescues brain abnormalities caused by Nf1 inactiviation

(A) Control and Nf1hGFAPCKO mice were treated with vehicle and MEKi from P0.5-P18 and analyzed at P18. (B) The body weight of treated control and mutant mice was quantified. (C) Triple labeling of p-Erk/Olig2/Ascl1 was performed on brain sections of vehicle-treated control and Nf1hGFAPCKO as well as MEKi-treated Nf1hGFAPCKO mice. Arrows and arrowheads: Olig2+/Ascl1+ and Olig2+/Ascl1- cells. Of note, p-Erk+/Olig2+/Ascl1+ cells were only identified in vehicle-treated mutants. (C’, C’’) The number of p-Erk+ cells and the percentage of Olig2+ cells among total SVZ Ascl1+ cells were quantified among vehicle and MEKi-treated control and mutant mice. (D) GFAP/Olig2 double-labeling of the CC from treated control and mutant mice was performed. Dashed lines mark the border between the CC and cerebral cortex (Ctx). (D’, D”) The thickness of the CC and the density of GFAP+ and Olig2+ cells in the CC of treated control and mutant mice were quantified. NeuN staining in the treated control and mutant OB is shown (E) and quantified (E’). Bottom panels in (E) are the higher magnification view of the boxed areas in the top panels. (F) Proposed model for the mechanism by which Nf1-regulated Erk signaling pathway regulates neuronal/glial fate specification of SVZ progenitors. SVZ progenitor cells most affected by Nf1 inactivation are labeled as red (see main text). All the quantification data are presented as mean ± SEM. Scale bars: 50 μm. See also Figure S8.