A missense point mutation in nerve growth factor (NGFR100W) results in selective peripheral sensory neuropathy

Abstract

The R100W mutation in nerve growth factor is associated with hereditary sensory autonomic neuropathy V in a Swedish family. These patients develop severe loss of perception to deep pain but with apparently normal cognitive functions. To better understand the disease mechanism, we examined a knockin mouse model of HSAN V. The homozygous mice showed significant structural deficits in intra-epidermal nerve fibers (IENFs) at birth. These mice had a total loss of pain perception at ∼2 months of age and often failed to survive to adulthood. Heterozygous mutant mice developed a progressive degeneration of small sensory fibers both behaviorally and functionally: they showed a progressive loss of IENFs starting at the age of 9 months accompanied with progressive loss of perception to painful stimuli such as noxious temperature. Quantitative analysis of lumbar 4/5 dorsal root ganglia revealed a significant reduction in small size neurons, while analysis of sciatic nerve fibers revealed the heterozygous mutant mice had no reduction in myelinated nerve fibers. Significantly, the amount of NGF secreted from mouse embryonic fibroblasts were reduced from both heterozygous and homozygous mice compared to their wild-type littermates. Interestingly, the heterozygous mice showed no apparent structural alteration in the brain: neither the anterior cingulate cortex nor the medial septum including NGF-dependent basal forebrain cholinergic neurons. Accordingly, these animals did not develop appreciable deficits in tests for brain function. Our study has thus demonstrated that the NGF^R100W mutation likely affects the structure and function of peripheral sensory neurons.

Keywords: Cognition; Dorsal root ganglion; Hereditary sensory autonomic neuropathy V; Intraepidermal nerve fiber; Knockin mouse model; Nerve growth factor; Nociception; Pain; TrkA; p75 neutrophic factor receptor.

Fig. 1.

The +/fln and fln/fln mutant mice exhibit loss of CGRP-positive neurons in DRGs. (A) The body weight of three genotype was monitored over a period of 8 weeks (+/+: n = 3; +/fln: n = 5; fln/fln: n = 3). (B) Pups of the three genotypes at postnatal 5 days. (C) Immunoreactivity to CGRP in representative sections of lumbar DRG from P0 mice. (D) Cell size distribution of CGRP-positive neurons in DRG. +/+: n = 3 (423 cells); +/fln n = 3 (431cells); fln/fln: n = 3 (341 cells). All p values were calculated using nonparametric Kolmogorov-Smirnov test. (E) Cumulative distribution of CGRP-positive neurons in DRG. *=p < 0.05, ****=p < 0.0001, one-way ANOVA test followed by Dunn’s multiple comparisons test (compared with +/+ group).

Fig. 2.

The fln/fln mutant mice display decreased responsiveness to pain at 2 months of age. (A) The fln/fln, not +/fln mutant mice showed significant deficits (20 s, cutoff time) in 55 °C hot plate assays at 2 months of age. Mean ± SEM, * = p < 0.05, ** = p < 0.01 by unpaired t test. +/+: n = 39; +/fln: n = 29; fln/fln: n = 2. (B) The fln/fln mutant mice showed serious hind paw damage after hot plate test.

Fig. 3.

The +/fln mice exhibit progressive loss of CGRP/TrkA-positive small sized DRG neurons. A: Images of representative sections of lumbar DRGs stained for CGRP/TrkA in +/+ and +/fln mice at 4 and 10 months of age. B: The quantitative results are shown. 3 pairs of mice (n = 3) were examined in each condition. The data were analyzed by t test.

Fig. 4.

The +/fln mice exhibit no loss of TrkB-positive large diameter DRG neurons. A: Images of representative sections of lumbar DRGs stained for CGRP/TrkB in +/+ and +/fln mice at 10 months of age; B: The quantitative results. 3 pairs of mice (n = 3) were examined in each conditions. The data were analyzed by t test.

Fig. 5.

The +/fln mice show no loss of myelinated fibers in sciatic nerve by P-Phenylene Diamine (PPD) staining. (A), (B), (C) Representative sections of the myelinated fibers in sciatic nerve from +/+ and +/fln mice of 2, 9 and 18 months of age respectively. Side panel was the size distribution of the myelinated fibers. One pair mice per group, 2 month (+/+, n = 2718 fibers; +/fln: n = 3682 fibers), 9 month (+/+: n = 1685 fibers; +/fln: n = 3524 fibers), 18 month (+/+: n = 1661 fibers; +/fln: n = 806 fibers). All p values were calculated using nonparametric Kolmogorov-Smirnov test.

Fig. 6.

The +/fln mutant mice exhibit progressive loss of PGP9.5-positive intra-epidermal sensory fibers (IENFs) and develop progressive sensory deficits. (A) Sections of footpad skin from 2-, 9- and 18 month-old mice were stained with antibodies to the pan-nerve fiber marker PGP9.5. (B) Quantification of PGP9.5-positive nerve fibers intensities in epidermis of footpad skin. To estimate the density of IENF in the epidermis, at least 12–15 images of each animal were analyzed throughout the section thickness and entire length of each image. About 2600 um of footskin per each animal was analyzed. epi: epidermis; d: dermis. Mean ± SEM, * = p < 0.05 by two-way ANOVA test. +/+: n = 3; +/fln: n = 3; (C) Hot plate assays at 2, 9 and 18 months of age. Mean ± SEM, * = p < 0.05 by two-way ANOVA. 2 month-old mice (+/+: n = 12; +/fln: n = 15), 9 month (+/+: n = 11; +/fln: n = 27), 18 month (+/+: n = 11; +/fln: n = 9).

Fig. 7.

The +/fln mutant mice show no significant deficit in Morris water maze test. (A) Diagram of Morris water maze. The test mice (18 month–old) were first trained to find a platform with a visible flag on days 1 to 3 and then a submerged hidden platform on days 4 to 7. The time (B) and path length (C) to find the platform was recorded. On day 8 of probe test, the platform was removed in session I. Times spent by mice in the target quadrant (D) and # passing of the target zone (E) were recorded. In session II, the visible platform was placed back and the latency time (F) was recorded. All data are presented as Mean ± SEM. p = 0.817 for B and p = 0.735 for C. p values were calculated by two-way ANOVA; p values for D, E, F by unpaired t test. +/+: n = 9, +/fln: n = 18.

Fig. 8.

NGF secretion is decreased in +/fln and fln/fln mouse embryonic fibroblasts. NGF protein content in media was measured by ELISA. Data from NGF ELISA was presented as Mean ± SEM; n = 6 for each experimental group. p values were calculated by Dunnett’s multiple comparisons test.