Central Alteration in Peripheral Neuropathy of Trembler-J Mice: Hippocampal pmp22 Expression and Behavioral Profile in Anxiety Tests

Abstract

Charcot-Marie-Tooth (CMT) type 1 disease is the most common human hereditary demyelinating neuropathy. Mutations in pmp22 cause about 70% of all CMT1. Trembler-J (TrJ/+) mice are an animal model of CMT1E, having the same spontaneous pmp22 mutation that is found in humans. We compared the behavior profile of TrJ/+ and +/+ (wild-type) in open-field and elevated-plus-maze anxiety tests. In these tests, TrJ/+ showed an exclusive head shake movement, a lower frequency of rearing, but a greater frequency of grooming. In elevated-plus-maze, TrJ/+ defecate more frequently, performed fewer total entries, and have fewer entries to closed arms. These hippocampus-associated behaviors in TrJ/+ are consistent with increased anxiety levels. The expression of pmp22 and soluble PMP22 were evaluated in E17-hippocampal neurons and adult hippocampus by in situ hybridization and successive immunohistochemistry. Likewise, the expression of pmp22 was confirmed by RT-qPCR in the entire isolated hippocampi of both genotypes. Moreover, the presence of aggregated PMP22 was evidenced in unmasked granular hippocampal adult neurons and shows genotypic differences. We showed for the first time a behavior profile trait associated with anxiety and a differential expression of pmp22/PMP22 in hippocampal neurons of TrJ/+ and +/+ mice, demonstrating the involvement at the central level in an animal model of peripheral neuropathy (CMT1E).

Keywords: CA3 neurons; Charcot–Marie–Tooth; Trembler-J; anxiety; hippocampus; peripheral-myelin-protein-22.

Figure 1

Movement parameters of +/+ mice and TrJ/+ mice in the open field test. (A) Latency. (B) Duration in the center zone. (C) Duration in the peripheral zone. (D) Distance moved. (E) TMLM: time during which the mice were making locomotion movement. (F) Velocity. Parameters in (A–C) were not parametrically distributed and they were analyzed using the Mann–Whitney U-test. Parameters in (D–F) were normally distributed and analyzed using Student’s t-test, df = 18. Asterisk indicates a significant difference between TrJ/+ (gray bars, n = 9) and +/+ (white bars, n = 11). ** p < 0.01, *** p < 0.001, **** p < 0.0001. The mean is shown as “+”.

Figure 2

Rearing, grooming, defecations, and head shake of +/+ mice and TrJ/+ mice. (A) Parameters analyzed in open field test. (B) Parameters analyzed in elevated plus maze test. Rearing and grooming behaviors were analyzed using Student’s t-test, df = 18. Defecations and head shake were analyzed using the Mann–Whitney U-test. Asterisk indicates significant difference between TrJ/+ (gray bars, n = 9) and +/+ (white bars, n = 11) mice: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The mean is shown as “+”.

Figure 3

Entries in open and closed arms of +/+ mice and TrJ/+ mice in the elevated plus maze test. The behavioral parameters total and closed arms were analyzed using Student’s t-test, and for open arms, was analyzed using the Mann–Whitney U-test. Asterisk indicates a significant difference between TrJ/+ (gray bars, n = 9), and +/+ (white bars, n = 11) mice: * p < 0.05, ** p < 0.01. The mean is shown as “+”.

Figure 4

In situ hybridization of pmp22 mRNA and associated PMP22 immunostaining in adult hippocampal CA3 neurons. (A) A panoramic view of the CA3 hippocampal regions show the ISH pmp22 signal (green) and their associated PMP22 (soluble form of protein) (magenta) in +/+ and TrJ/+ genotypes. (B) In situ hybridization of pmp22 mRNA in hippocampal adult CA3 neurons of +/+ and TrJ/+ mice brains, showing the pmp22 transcript was detected in +/+ and TrJ/+ brain cryosections at the hippocampal CA3 region. The hybrid signals were observed in nuclear and perinuclear domains. Moreover, correlative post-ISH immunostaining of PMP22 and NF-68 may be observed. The upper panel shows +/+ and the lower panel shows the TrJ/+ fluorescence intensity of hybrid and proteins. (C) Hippocampal pmp22 mRNA expression in +/+ y TrJ/+ mice was determined by RT-qPCR. Comparative analysis between +/+ and TrJ/+ hippocampi shows no significant differences (Student’s t-test, p = 0.62, n = 3 for each genotype). mRNA levels were normalized against β-actin mRNA. “+”: mean. The mean is shown as “+”. Scale bar for A = 50 µm for all panels. Scale bar for B = 5 μm for all panels.

Figure 5

Localization of aggregated and soluble PMP22 in the hippocampus of adult mice. (A) A tail scan image of the whole hippocampal formation of both +/+ and TrJ/+ genotypes. The asterisk (*) indicates hippocampal CA3 neurons regions. Nuclear domains were enlightened by specific DAPI staining. Expression of PMP22 (magenta) aggregates in the hippocampus after epitope retrieval (70% formic acid), in 60 μm thick vibratome sections of both +/+ and TrJ/+. (B) PMP22 aggregated is shown in the CA3 pyramidal neurons of both +/+ and TrJ/+ mice. (C) Specific PMP22 showed significant differences between genotypes, with the highest values observed in TrJ/+ mice, analyzed by the Mann–Whitney U-test (p < 0.0001, n = 4 for each genotype). Scale bar for A = 200 μm for all panels. Scale for B = 10 μm for all panels. **** p < 0.0001. The mean is shown as “+”.