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. 2024 Jan 23;16(3):477.
doi: 10.3390/cancers16030477.

Gender-Specific Fine Motor Skill Learning Is Impaired by Myelin-Targeted Neurofibromatosis Type 1 Gene Mutation

Daniella P Hernandez  1 , Daniela M Cruz  1 , Celeste S Martinez  1 , Larisa M Garcia  1 , Ashley Figueroa  1 , Marisol Villarreal  1 , Liya M Manoj  1 , Saul Lopez  1 , Karla D López-Lorenzo  2 , Alejandro López-Juárez  1
Affiliations

Gender-Specific Fine Motor Skill Learning Is Impaired by Myelin-Targeted Neurofibromatosis Type 1 Gene Mutation

Daniella P Hernandez et al. Cancers (Basel). .

Abstract

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene. The clinical presentation of NF1 includes diverse neurological issues in pediatric and adult patients, ranging from learning disabilities, motor skill issues, and attention deficit disorder, to increased risk of depression and dementia. Preclinical research suggests that abnormal neuronal signaling mediates spatial learning and attention issues in NF1; however, drugs that improve phenotypes in models show inconclusive results in clinical trials, highlighting the need for a better understanding of NF1 pathophysiology and broader therapeutic options. Most NF1 patients show abnormalities in their brain white matter (WM) and myelin, and links with NF1 neuropathophysiology have been suggested; however, no current data can clearly support or refute this idea. We reported that myelin-targeted Nf1 mutation impacts oligodendrocyte signaling, myelin ultrastructure, WM connectivity, and sensory-motor behaviors in mice; however, any impact on learning and memory remains unknown. Here, we adapted a voluntary running test-the complex wheel (CW; a wheel with unevenly spaced rungs)-to delineate fine motor skill learning curves following induction of an Nf1 mutation in pre-existing myelinating cells (pNf1 mice). We found that pNf1 mutant females experience delayed or impaired learning in the CW, while proper learning in pNf1 males is predominantly disrupted; these phenotypes add complexity to the gender-dependent learning differences in the mouse strain used. No broad differences in memory of acquired CW skills were detected in any gender, but gene-dose effects were observed at the studied time points. Finally, nitric oxide signaling regulation differentially impacted learning in wild type (WT)/pNf1, male/female mice. Our results provide evidence for fine motor skill learning issues upon induction of an Nf1 mutation in mature myelinating cells. Together with previous connectivity, cellular, and molecular analyses, these results diversify the potential treatments for neurological issues in NF1.

Keywords: RASopathies; myelin; neurofibromatosis type 1; oligodendrocytes; white matter.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Gender-dependent differences in CW performance in WT mice. X axis labels are shared in (B–F). (A) CW learning/memory test: individual mice were introduced to cages with CWs for 2 weeks (blue, left), housed without wheels for 3 weeks (gap), and re-introduced to CWs for 1 additional week (blue, right). (B–F) Plots for nightly (12 h dark period) values of CW parameters for wild-type (WT) female (black) and male (green) mice: (B) total distance run (meters), (C) Total Average Speed (TAS; average speed of all minutes per night), (D) max speed (maximum speed in meters/minute achieved per night), (E) Activity Average Speed (AAS, average speed of minutes running >1 m), and (F) activity (number of minutes running >1 m). Only statistically significant p values (two-way ANOVA test, gender as source of variation, females n = 11 and males n = 5) are shown under the plots for the first (nights 1–14, left) or second (nights 36–42, right) introduction to CWs. Comparison of individual nights in female vs. male are also shown (Bonferroni’s post hoc tests, * p < 0.05). Insets: statistical comparison of slopes and intercept/elevation of slopes (* p < 0.05 and # p < 0.05, respectively, two-tailed linear regression) between female and male mice for the first introduction to CWs.
Figure 2
Figure 2
pNf1 mutation in females delays fine motor skill acquisition. X axis labels are shared in (B–F). (A) Experimental protocol: 2-month-old (2 MO) WT, pNf1f/+, and pNf1f/f mice were treated with tamoxifen and 2–6 months later were introduced to CWs for 2 weeks (blue, left), housed without CW for 3 weeks (gap), and re-introduced to CWs for 1 week (blue, right). (B–F) Nightly values for CW parameters for wild-type (black), pNf1f/+ (red), and pNf1f/f (blue) mice are plotted: (B) total distance run, (C) Total Average Speed (TAS), (D) max speed, (E) Activity Average Speed (AAS), and (F) activity. Significant p values from two-way ANOVA tests (genotype as source of variation; WT n = 11; pNf1f/+ n = 6; pNf1f/f n = 6) are shown under plots for the first (nights 1–14, left) or second (nights 36–42, right) introduction to CWs. Individual night comparisons for WT vs. pNf1f/+ and vs. pNf1f/f (Bonferroni’s post hoc tests, * p < 0.05) are shown. Insets: comparison of slopes and intercept/elevation of slopes for WT vs. pNf1f/+ and vs. pNf1f/f are shown (two-tailed linear regression, * p < 0.05, and # p < 0.05, respectively).
Figure 3
Figure 3
pNf1 mutation in males impairs fine motor skill acquisition. X axis labels are shared in (A–E). (A–E) Nightly values for CW parameters for wild-type (green), pNf1f/+ (red), and pNf1f/f (blue) mice are plotted: (A) total distance run, (B) Total Average Speed (TAS), (C) max speed, (D) Activity Average Speed (AAS), and (E) activity. Significant p values from two-way ANOVA tests (genotype as source of variation; WT n = 5; pNf1f/+ n = 9; pNf1f/f n = 6) are shown under or above plots for the first (nights 1–14, left) or second (nights 36–42, right) introduction to CWs. Individual night comparisons for WT vs. pNf1f/+ and WT vs. pNf1f/f (Bonferroni’s post hoc tests, * p < 0.05) are shown. Insets: comparison of slopes and intercept/elevation of slopes for WT vs. pNf1f/+ and WT vs. pNf1f/f are shown (two-tailed linear regression, * p < 0.05 and # p < 0.05, respectively).
Figure 4
Figure 4
Recombination driven by PlpCreER is highly specific for OL lineage cells in the adult hippocampus. Representative images of brain sections containing the hippocampus of WT (A,D), pNf1f/+ (B), and pNf1f/f (C,E) mice subjected to CW tests. Sections were immunostained to detect the reporter EGFP (green), the OL-lineage marker Olig2 (red), and the neuronal marker NeuN (white). Cell nuclei are labeled with DAPI (blue). Arrows indicate EGFP+ recombinant cells that are also Olig2+ but not NeuN+. (D,E) High magnification orthogonal projection (Z axis indicated at bottom right) of areas in (A,C) (dotted line) depicting EGFP+Olig2+ cells in close proximity to, but independently from, NeuN+ cells. (A–C) scale bar = 50 μm, (D,E) scale bar = 20 μm.
Figure 5
Figure 5
Number/fate of recombined cells is comparable in the CC of WT and pNf1 mice subjected to CW tests. X axis labels shared in H, K, and I, L. Representative images from female (A–C) and male (D–F) brain sections containing the CC ((G), midline region) of WT (A,D), pNf1f/+ (B,E), and pNf1f/f (C,F) mice subjected to CW tests. Sections were immunostained to detect the reporter EGFP (green), the oligodendrocyte progenitor (OPC) marker NG2 (red), and the OL marker CC1 (white). Cell nuclei are labeled with DAPI (blue) and non-specific CC1 signals in some blood vessels are indicated with a “v” (cyan). Pink arrows show NG2+EGFP- non-recombinant OPCs in close interaction with OLs. White arrows indicate EGFP+ recombinant cells co-localizing with CC1 but not with NG2. Scale bar = 25 μm. Cell quantification of all DAPI+ cells (H), % of EGFP+ recombinant cells/DAPI+ cells (I), CC1+ OLs/DAPI+ cells (J), EGFP+CC1+ recombinant OLs/DAPI+ cells (K), and EGFP+CC1+ recombinant OLs/recombinant cells (L). Student’s t test: * p < 0.05 (n = 4 mice/genotype/gender). Data are shown as the mean + SEM.
Figure 6
Figure 6
Progressive and gene-dose-dependent CW running issues from N1 and N7 in pNf1 mutant females. X axis labels are shared in (A–E). (A–E) Data from night 1 (N1, left) and night 7 (N7, right) of the first introduction to CWs were divided into 15 min intervals and CW parameters for wild-type (black), pNf1f/+ (red), and pNf1f/f (blue) mice were plotted: (A) total distance run, (B) Total Average Speed (TAS), (C) max speed, (D) Activity Average Speed (AAS), and (E) activity. Significant p values from two-way ANOVA tests with genotype as the source of variation are shown for each plot. Individual night comparisons for WT vs. pNf1f/+ and WT vs. pNf1f/f (Bonferroni’s post hoc tests, * p < 0.05) are shown.
Figure 7
Figure 7
L-NAME treatment modifies learning curves in pNf1 and WT females. X axis labels are shared in (A–E). Plots for nightly values of CW parameters for WT (black dotted line) and pNf1 f/+ (red dotted line) female mice treated with L-NAME (0.3 mg/L in drinking water): (A) total distance (meters), (B) Total Average Speed (TAS), (C) max speed, (D) Activity Average Speed (AAS), and (E) minutes with activity. Plots for untreated WT and pNf1 females are shown in faded colors as control values (compare with Figure 2). Statistically significant p values (two-way ANOVA test, treatment as source of variation) are shown under the plots for first (left) and second (right) introduction to CWs (* p red: L-NAME-treated pNf1 f/+ vs. untreated WT; * p black: L-NAME-treated WTs vs. untreated WTs). No differences between untreated vs. L-NAME-treated pNf1 f/+ mice were found. Comparison of individual nights are also shown (same color code, Bonferroni’s post hoc tests, * p < 0.05).
Figure 8
Figure 8
Working hypothesis for CW motor learning issues following Nf1 mutation induction in myelinating cells. (A) Hemizygous (red) or homozygous (blue) Nf1 mutation in myelinating cells (pNf1 mice) increases RAS/MAPK, nitric oxide (NO [45])1, and Notch [44]2 signaling ~6 months (slow) or ~1 month (fast) post-mutation induction. Myelin decompaction (right) and non-cell-autonomous (top) defects are downstream effects. (B) Learning curves of fine motor skills (voluntary CW running) show gender-dependent differences in WT mice (left). Additionally, an Nf1 mutation in myelin affects learning curves in both female and male pNf1 mice, with a modest influence of the Nf1 gene dose (right, red vs. blue lines). Defective NO signaling regulates CW learning in WT and pNf1 mice by unknown mediators (red dotted arrow). Abnormal downstream Notch signaling and myelin decompaction (gray dotted arrows) might contribute to motor learning issues.
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