Sex Is a major determinant of neuronal dysfunction in neurofibromatosis type 1

Abstract

Objective: Children with neurofibromatosis-1 (NF1) are at risk for developing numerous nervous system abnormalities, including cognitive problems and brain tumors (optic pathway glioma). Currently, there are few prognostic factors that predict clinical manifestations or outcomes in patients, even in families with an identical NF1 gene mutation. In this study, we leveraged Nf1 genetically engineered mice (GEM) to define the potential role of sex as a clinically relevant modifier of NF1-associated neuronal dysfunction.

Methods: Deidentified clinical data were analyzed to determine the impact of sex on optic glioma-associated visual decline in children with NF1. In addition, Nf1 GEM were employed as experimental platforms to investigate sexually dimorphic differences in learning/memory, visual acuity, retinal ganglion cell (RGC) death, and Nf1 protein (neurofibromin)-regulated signaling pathway function (Ras activity, cyclic adenosine monophosphate [cAMP], and dopamine levels).

Results: Female patients with NF1-associated optic glioma were twice as likely to undergo brain magnetic resonance imaging for visual symptoms and 3× more likely to require treatment for visual decline than their male counterparts. As such, only female Nf1 GEM exhibited a decrement in optic glioma-associated visual acuity, shorter RGC axons, and attenuated cAMP levels. In contrast, only male Nf1 GEM showed spatial learning/memory deficits, increased Ras activity, and reduced dopamine levels.

Interpretation: Collectively, these observations establish sex as a major prognostic factor underlying neuronal dysfunction in NF1, and suggest that sex should be considered when interpreting future preclinical and clinical study results.

Figure 1. Optic glioma location and size is not impacted by sex

(A) Girls with NF1-associated optic glioma were 3 times more likely to be treated for visual decline than their male counterparts (p=0.0007). (B) Indications for brain MRI. *Other includes one individual each imaged for weakness and the presence of a facial port-wine stain. (C) Locations of optic pathway gliomas (OPGs) in male and female children with NF1 occur at similar frequencies. (D) Frequency of visual decline necessitating treatment in girls and boys stratified by tumor location.

Figure 2. Optic glioma-associated vision disturbances are greater in girls with NF1 and in female Nf1-CKO mice

(A) Optic nerve volumes in Nf1-CKO mice (black bars) are larger than those observed in control littermate mice (white bars), regardless of sex. Asterisks denotes p<0.05. (B) Only female Nf1-CKO mice have impaired visual acuity (VOS; cycles/degree) relative to controls. (C) Greater retinal ganglion cell apoptosis (%TUNEL+ cells) was observed in female Nf1-CKO mice (11.5-fold over female controls) relative to Nf1-CKO males (6.2-fold over male controls). (D) Representative images of retinal ganglion cells in culture demonstrate that female, but not male, Nf1-CKO neurons have reduced axon lengths relative to controls. (E) Female, but not male, Nf1-CKO neurons exhibit shorter axon lengths (~50% reduction) relative to controls. Open (white) bars denote control (CTL) mice; closed (black) bars denote Nf1-CKO mice. *p<0.05, **p<0.01.

Figure 3. Spatial learning impairments in the Morris Water Maze occur only in Nf1-CKO male mice

(A-B) During the first (A) and second (B) probe trials, control mice and female Nf1-CKO mice spent significantly more time in the target quadrant than all the other quadrants. In contrast, male Nf1-CKO mice showed no spatial preference and spent equal time in all quadrants. (C-D) Time spent in the target quadrant during the first (C) and second (D) probe trials was reduced by 25% and 23%, respectively in male Nf1-CKO mice. (E-F) Male Nf1-CKO mice entered the target 1.5-fold less frequently than controls in both the first (E) and second (F) probe trials. *p<0.05, **p<0.01.

Figure 4. Sexually dimorphic regulation of neurofibromin signaling pathways

(A) Established molecular pathways regulated by the NF1 gene product, neurofibromin. (B) Retinal cyclic adenosine monophosphate (cAMP) levels were reduced by 47% in female Nf1-CKO mice relative to female controls. No change in retinal cAMP levels was observed in male Nf1-CKO mice relative to male controls. (C) Hippocampal dopamine levels were reduced 2-fold in male Nf1-CKO mice compared to male controls. No differences were observed in female Nf1-CKO mice relative to female controls. (D) DARPP32 phosphorylation was reduced in the hippocampus of Nf1-CKO male, but not female, mice. (E, F) Densitometric quantification reveals increased active (guanosine triphosphate [GTP]-bound) Ras (Ras-GTP levels relative to total Ras; E) and ERK (phospho-ERK1/2 relative to total ERK1/2; F) activation in male, but not female, Nf1-CKO hippocampi relative to controls. *p <0.05.