Elucidating the impact of neurofibromatosis-1 germline mutations on neurofibromin function and dopamine-based learning

Abstract

Neurofibromatosis type 1 (NF1) is a common autosomal dominant neurologic condition characterized by significant clinical heterogeneity, ranging from malignant cancers to cognitive deficits. Recent studies have begun to reveal rare genotype-phenotype correlations, suggesting that the specific germline NF1 gene mutation may be one factor underlying disease heterogeneity. The purpose of this study was to define the impact of the germline NF1 gene mutation on brain neurofibromin function relevant to learning. Herein, we employ human NF1-patient primary skin fibroblasts, induced pluripotent stem cells and derivative neural progenitor cells (NPCs) to demonstrate that NF1 germline mutations have dramatic effects on neurofibromin expression. Moreover, while all NF1-patient NPCs exhibit increased RAS activation and reduced cyclic AMP generation, there was a neurofibromin dose-dependent reduction in dopamine (DA) levels. Additionally, we leveraged two complementary Nf1 genetically-engineered mouse strains in which hippocampal-based learning and memory is DA-dependent to establish that neuronal DA levels and signaling as well as mouse spatial learning are controlled in an Nf1 gene dose-dependent manner. Collectively, this is the first demonstration that different germline NF1 gene mutations differentially dictate neurofibromin function in the brain.

Figure 1.

Germline NF1 gene mutations result in different levels of neurofibromin expression in NF1-patient fibroblasts. (A) Neurofibromin immunoblot of NF1-patient fibroblasts reveals two groups; Group 1 patients have <25% neurofibromin expression (n = 4) and Group 2 patients have >70% reductions in neurofibromin expression (n = 7 shown) compared with sex- and age-matched controls. Tubulin was used as an internal protein loading control. (B) RAS activity is significantly elevated in all NF1-patient fibroblasts. (C) Schematic representation of the NF1 gene with the identified NF1-patient mutations and summary of NF1-patient germline NF1 gene mutations, demographic information and genotyping results. The number of the exon mutated is indicated above the mutation location. Light and dark boxes indicate alternate exons. Horizontal bars represent the length of the predicted transcribed mRNA. TGD, total genomic deletion; FS, frame shift mutation. All data are represented as means ± SEM (*P < 0.0001, one-way ANOVA with Bonferroni post-test).

Figure 2.

Germline NF1 gene mutations promote differential neurofibromin expression in NF1-patient-derived iPSCs. (A) Immunostaining of the iPSC cultures with stem cell-specific antibodies confirmed the pluripotent nature of the cultured iPSCs. Scale bar, 50 µm. (B) Bright-field imaging of representative iPSCs. Scale bar, 100 µm. (C) Neurofibromin expression is only significantly reduced in group 2 NF1-patient-derived iPSCs. (D) All NF1-patient-derived iPSCs have elevated RAS activity levels (RAS-GTP) relative to control iPSC lines. All data are represented as means ± SEM (*P < 0.0001, one-way ANOVA with Bonferroni post-test).

Figure 3.

Germline NF1 gene mutations promote differential neurofibromin expression in NF1-patient-derived NPCs. (A) Bright field imaging and immunostaining of the NPC cultures with the SMI-312 pan-axonal (neuronal) antibody. Scale bar left panel, 100 µm; Scale bar right panel, 50 µm. (B) Neurofibromin expression is only significantly different in group 2 NF1-patient samples compared with control NPCs. (C) RAS activity is significantly elevated in all NF1-patient NPCs. (D) cAMP is decreased in all NF1-NPCs compared with control NPCs.

Figure 4.

Germline NF1 gene mutations regulate DA signaling in a gene dose-dependent manner in NF1-patient NPCs. (A) DA levels are reduced in group 2 NF1-patient samples compared with control NPCs. (B) Correlation of relative neurofibromin expression and DA levels. (C) DARPP-32 activity (phosphorylation) is only reduced in group 2 NF1-patient samples compared with control NPCs. (D) Correlation of relative neurofibromin expression and DARPP-32 phosphorylation. All data are represented as means ± SEM (*P < 0.0001, one-way ANOVA with Bonferroni post-test).

Figure 5.

Mouse learning is regulated in an Nf1 gene dose-dependent manner. (A) Both Nf1+/− and Nf1+/− (WT) mice preferentially occupy the target quadrant and exhibit equivalent occupancies in the target quadrant during probe trial 2 of the Morris water maze test. Dashed line depicts chance occupancy of a quadrant in the water maze. (B) Neurofibromin expression is reduced by 54% in Nf1+/− mice and by 85% in Nf1^FMC mice. (C) Both Nf1^FFC and Nf1^FF (control) mice preferentially occupy the target quadrant. Null Nf1^FFC mice spent significantly less time in the target quadrant relative to controls. (D) Neurofibromin expression is reduced by 50% in Nf1^FWC mice and by 80% in Nf1^FFC mice compared with controls. (E) Extensive recombination of GFAP-Cre (green) in NeuN⁺ neurons (red) is depicted by immunofluorescent staining within the CA1, CA2, CA3 and dentate gyrus (DG) of the hippocampus of RosaGREEN^GFAP-Cre mice. Scale bars, 50 µm. All data are represented as means ± SEM (***P < 0.0001; **P < 0.001; *P < 0.01, one-way/two-way ANOVA with Bonferroni post-test).

Figure 6.

Mouse DA signaling and learning is regulated in an Nf1 gene dose-dependent manner. (A and B) DA is reduced in gene dose-dependent manner in heterozygous (Nf1+/− and Nf1^FWC) and null (Nf1^FMC and Nf1^FFC) mice compared with controls. (C) Correlation between neurofibromin expression and DA levels. (D and E) DARPP-32 phosphorylation is reduced in a neurofibromin dose-dependent manner. (F) Neurofibromin expression highly correlates with pDARPP32 levels in mouse hippocampi. All data are represented as means ± SEM (***P < 0.0001; **P < 0.001; *P < 0.01, one-way ANOVA with Bonferroni post-test).

Figure 7.

Neurofibromin regulates dopaminergic neurons in a gene dose-dependent manner. (A) TH-Cre (green) recombination occurs exclusively in TH⁺ dopaminergic neurons (red) in the VTA (top) of Rosa-GREEN^TH-Cre mice. Scale bar, 50 µm. (B) All Nf1^TH mice exhibited preferential occupancy for the target quadrant. Nf1^TH−/− mice exhibited reduced quadrant occupancy in the target quadrant relative to Nf1^TH+/− and Nf1^TH+/+ mice during probe trial 2 of the Morris water maze test. Dashed line depicts chance occupancy of a quadrant in the water maze. (C) Neurofibromin expression is reduced by 42% in Nf1^TH+/− mice and by 72% in Nf1^TH−/− mice compared with controls. (D) Hippocampus DA levels are reduced by 38% in Nf1^TH+/− mice and by 69% in Nf1^TH−/− mice compared with controls. (E) Western blots (left) and quantification (right) of DARPP-32 Thr34 phosphorylation (n = 5 mice per genotype). All data are represented as means ± SEM (***P < 0.0001; **P < 0.05; *P < 0.01, one-way/ two-way ANOVA with Bonferroni post-test).