Neurofilament light polypeptide gene N98S mutation in mice leads to neurofilament network abnormalities and a Charcot-Marie-Tooth Type 2E phenotype

Abstract

Charcot-Marie-Tooth disease (CMT) is the most commonly inherited neurological disorder with a prevalence of 1 in 2500 people worldwide. Patients suffer from degeneration of the peripheral nerves that control sensory information of the foot/leg and hand/arm. Multiple mutations in the neurofilament light polypeptide gene, NEFL, cause CMT2E. Previous studies in transfected cells showed that expression of disease-associated neurofilament light chain variants results in abnormal intermediate filament networks associated with defects in axonal transport. We have now generated knock-in mice with two different point mutations in Nefl: P8R that has been reported in multiple families with variable age of onset and N98S that has been described as an early-onset, sporadic mutation in multiple individuals. Nefl(P8R/+) and Nefl(P8R/P8R) mice were indistinguishable from Nefl(+/+) in terms of behavioral phenotype. In contrast, Nefl(N98S/+) mice had a noticeable tremor, and most animals showed a hindlimb clasping phenotype. Immunohistochemical analysis revealed multiple inclusions in the cell bodies and proximal axons of spinal cord neurons, disorganized processes in the cerebellum and abnormal processes in the cerebral cortex and pons. Abnormal processes were observed as early as post-natal day 7. Electron microscopic analysis of sciatic nerves showed a reduction in the number of neurofilaments, an increase in the number of microtubules and a decrease in the axonal diameters. The Nefl(N98S/+) mice provide an excellent model to study the pathogenesis of CMT2E and should prove useful for testing potential therapies.

Figure 1.

Generation of Nefl P8R and N98S knock-in mice. (A) Strategy for generating the knock-in mice: a targeting vector containing the point mutation in the first exon of Nefl, and a Loxp-Neo-Loxp cassette inserted in the first intron was constructed and used to generate the knock-in mice. The targeted allele could be differentiated from the WT allele by PCR using the 4F and 4R primers. (B) Ethidium bromide-stained agarose gel of PCR analyses of the F1 mice showing the WT and targeted (L83 (N98S)) alleles; five of the nine animals showed both alleles, the other four were WT. (C) RNA was isolated from brains and spinal cords of Nefl^+/+, Nefl^P8R/+ Nefl^P8R/P8R and Nefl^N98S/+ mice and the amount of NFL mRNA was quantified by RT–PCR and compared with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. Ratios of NFL mRNA and GAPDH mRNA for the different genotypes are shown. Experiments were repeated with three sets of animals. (D) Western blots of brain, spinal cord and sciatic nerve from Nefl^+/+, Nefl^P8R/+, Nefl^P8R/P8R and Nefl^N98S/+ probed with anti-NFL-N antibody and anti-GAPDH. (E) Ratios of NFL/GAPDH from the western blots shown in D. (Brain: Nefl^+/+: n = 10; Nefl^P8R/+: n = 9; Nefl^P8R/P8R: n = 10; Nefl^N98S/+: n = 9; Spinal cord: Nefl^+/+: n = 10; Nefl^P8R/+: n = 9; Nefl^P8R/P8R: n = 10; Nefl^N98S/+: n = 9; Sciatic nerve: Nefl^+/+: n = 9; Nefl^P8R/+: n = 8; Nefl^P8R/P8R: n = 9; Nefl^N98S/+: n = 7). Significance was calculated using a one-tailed, type 3 t-test in excel (*P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001).

Figure 2.

Abnormal hindlimb posturing and tremor in Nefl^N98S/+ mice. (A) Photograph of Nefl^+/+ mouse showing normal hindlimb posturing when suspended by its tail. (B) Photograph of Nefl^N98S/+ mouse showing abnormal hindlimb posturing (clasping) when suspended by its tail. (C) Percentages of Nefl^N98S/+ mice (blue bars) and Nefl^+/+ (WT) mice (red bars) that were shaking in different age groups. (D) Percentages of Nefl^N98S/+ mice (blue bars) and Nefl^+/+ (WT) mice (red bars) demonstrating hindlimb clasping in different age groups. (E) Blue bars: percentage of mice that were shaking before backcrossing to C57Bl6 mice five times; red bars: percentage of mice that were shaking after backcrossing five times. The proportion of Nefl^N98S/+ mice that exhibited shaking increased after backcrossing. (F) Red bars: percentage of mice showing hindlimb clasping before backcrossing to C57Bl6 mice five times; red bars: percentage of mice that show hindlimb clasping after backcrossing. (Nefl^N98S/+: before backcrossing n = 71, after backcrossing n = 33; Nefl^+/+: before backcrossing n = 179; after backcrossing, n = 52).

Figure 3.

Immunofluorescence micrographs of sections of spinal cord stained with anti-NFL-N antibody showing inclusions in Nefl^N98S/+ mice. (A) Section of anterior horn of 18-month-old Nefl^+/+ (WT) mouse. (B) Section of anterior horn of 18-month-old Nefl^N98S/+ (N98S) mouse. (C) Section of posterior horn of 18-month-old Nefl^+/+ (WT) mouse. (D) Section of posterior horn of 18-month-old mutant Nefl^N98S/+ (N98S) mouse. WM of Nefl^+/+ mice shows axonal labeling, whereas there is relatively little labeling in the cell bodies of the GM in both the anterior and posterior horns. In contrast, there was relatively little labeling in the WM of Nefl^N98S/+ mice, except for the inclusions (arrow), which are likely due to axonal swellings. Inclusions are also observed in the GM in both the anterior and posterior sections (arrows). Bars = 50 μm.

Figure 4.

Immunofluorescence of sections of dorsal root ganglia showing abnormalities in Nefl^N98S/+ mice. (A and C) Labeling of dorsal root ganglia of WT mice; (B and D) Labeling of dorsal root ganglia of Nefl^N98S/+ mice. The cell bodies of the DRGs are stained with anti-NFL, and aggregates in the cell bodies can clearly be seen in the sections from the Nefl^N98S/+ mice. The processes from the Nefl^N98S/+ mice show a discontinuous staining with apparent aggregates. Bars (A and B) = 100 µm; (C and D) = 25 µm.

Figure 5.

Immunofluorescence micrographs of sections of cerebellum showing abnormalities in Nefl^N98S/+ mice. (A) Labeling of the cerebellum of 18-month-old Nefl^+/+ (WT) mouse with anti-NFL-N antibody. (B) Labeling of the cerebellum of 18-month-old Nefl^N98S/+ (N98S) mouse with anti-NFL-N antibody. Note labeling of the WM tract in the Nefl^+/+ mouse compared with the irregular labeling and the absence of the WM tract in the cerebellum of the Nefl^N98S/+ mouse (arrows in A and B). There are also abnormal processes in the granular layer (GL) in the cerebellum of the Nefl^N98S/+ mouse. (C and D) Double labeling of cerebellar section from 18-month old Nefl^+/+ mouse with anti-NFL-N (green) and anti-calbindin (red) antibodies. (E and F) Double labeling of cerebellar section from 18-month-old Nefl^+/+ mouse with anti-NFL-N (green) and anti-calbindin (red) antibodies. Arrows indicate labeling of basket cell axons around the Purkinje cells in WT mice (C). In Nefl^N98S/+ mice, note the absence of basket cell process labeling (arrow) and the swellings in the GL of the cerebellum (E). The absence of calbindin labeling (F) suggests that they are not Purkinje cell processes but rather disordered basket cell processes. Bars = 50 μm.

Figure 6.

Immunofluorescence micrographs showing labeling of a post-natal day 7 spinal cords of Nefl^+/+ (WT) and Nefl^N98S/+ (N98S) with anti-NFL-N antibody. (A) Section of anterior horn from Nefl^+/+ mouse. (B) Section of anterior horn from Nefl^N98S/+ mouse. (C) Section of posterior horn from Nefl^+/+ mouse. (D) Section of posterior horn from Nefl^N98S/+ mouse. Arrows in A and B indicate the abundant axonal labeling of the WM of the Nefl^+/+ sections, which is reduced in the Nefl^N98S/+ sections. In the GM, axonal tracts can be observed in Nefl^+/+ sections (arrows, C), whereas prominent cell body labeling is observed in the posterior horn section from the Nefl^N98S/+ mouse (arrows, D). Bars = 50 μm.

Figure 7.

Immunofluorescence micrographs showing labeling with anti-NFL-N antibody of sections of cerebella from Nefl^+/+ and Nefl^N98S/+ mice of various ages. (A) Section of cerebellum from a 7-day-old Nefl^+/+ (WT) mouse. (B) Section of cerebellum from a 7-day-old Nefl^N98S/+ (N98S) mouse. (C) Section of cerebellum from a 1-month-old Nefl^N98S/+ (N98S) mouse. (D) Section of cerebellum from a 4-month-old Nefl^N98S/+ (N98S) mouse. GL, granular layer. Arrows point to the white matter tracts. Bar = 50 μm.

Figure 8.

Electron micrographs of sciatic nerves from Nefl^+/+ and Nefl^N98S/+ mice. (A and B) Low power views of section of sciatic nerves from Nefl^+/+ mouse (A, WT) and Nefl^N98S/+ mouse (B, N98S). (C and D). High power views of sciatic nerves from Nefl^+/+ mouse (C, WT) and Nefl^N98S/+ mouse (D, N982). At low power (A and B), differences in axon diameter can be observed. At high power (C and D), neurofilaments can be seen in the section from the Nefl^+/+ mouse (C, shown in cross-section) that are missing in the sections from the Nefl^N98S/+ mouse, where they are replaced with microtubules (D). Three sets of animals were analyzed. Bars A and B = 2 μm; C and D = 100 μm.

Figure 9.

Axon size distribution and g-ratios based on the electron micrographs. (A) G ratio = fiber area/axon area. Axon and fiber areas were measured using the magic wand tool set to scale in ImageJ. (B) Nefl^+/+ and Nefl^P8R/+ mice show similar distribution of axon sizes, with axons >9 µm² occupying the greatest percentage of the sample measured. Nefl^N98S/+ mice show a different pattern of axon size distribution, with a greater percentage of axons with smaller areas. (C) Cumulative distribution of axon areas measured in Nefl^+/+ and Nefl^P8R/+ mice. The distributions are not significantly different (Kolmogorov–Smirnov KS test P > 0.05). (D) Cumulative distribution of axon areas measured in Nefl^+/+ and Nefl^N98S/+ mice. The distributions are significantly different (Kolmogorov–Smirnov KS test P < 0.001). (E) Nefl^+/+ and Nefl^P8R/+ mice show similar g-ratios axon sizes. Nefl^N98S/+ mice show a much higher g-ratio, indicating that there is more myelin on the axons of the Nefl^N98S/+ mutant mice; Nefl^+/+ n = 215, Nefl^N98S/+ n = 192, and Nefl^P8R/+ n = 100. Data were pooled from the results from three different mice.