Reduced mitochondrial size in hippocampus and psychiatric behavioral changes in the mutant mice with homologous mutation of Timm8a1-I23fs49X

Abstract

Background: Deafness-dystonia-optic neuronopathy (DDON) syndrome, a condition that predominantly affects males, is caused by mutations in translocase of mitochondrial inner membrane 8A (TIMM8A)/deafness dystonia protein 1 (DDP1) gene and characterized by progressive deafness coupled with other neurological abnormalities. In a previous study, we demonstrated the phenotype of male mice carrying the hemizygous mutation of Timm8a1-I23fs49X. In a follow-up to that study, this study aimed to observe the behavioral changes in the female mutant (MUT) mice with homologous mutation of Timm8a1 and to elucidate the underlying mechanism for the behavioral changes.

Materials and methods: Histological analysis, transmission electron microscopy (EM), Western blotting, hearing measurement by auditory brainstem response (ABR), and behavioral observation were compared between the MUT mice and wild-type (WT) littermates.

Results: The weight of the female MUT mice was less than that of the WT mice. Among MUT mice, both male and female mice showed hearing impairment, anxiety-like behavior by the elevated plus maze test, and cognitive deficit by the Morris water maze test. Furthermore, the female MUT mice exhibited coordination problems in the balance beam test. Although the general neuronal loss was not found in the hippocampus of the MUT genotype, EM assessment indicated that the mitochondrial size showing as aspect ratio and form factor in the hippocampus of the MUT strain was significantly reduced compared to that in the WT genotype. More importantly, this phenomenon was correlated with the upregulation of translation of mitochondrial fission process protein 1(Mtfp1)/mitochondrial 18 kDa protein (Mtp18), a key fission factor that is a positive regulator of mitochondrial fission and mitochondrial size. Interestingly, significant reductions in the size of the uterus and ovaries were noted in the female MUT mice, which contributed to significantly lower fertility in the MUT mice.

Conclusion: Together, a homologous mutation in the Timm8a1 gene caused the hearing impairment and psychiatric behavioral changes in the MUT mice; the latter phenotype might be related to a reduction in mitochondrial size regulated by MTP18.

Keywords: DDP1; MTFP1; TIMM8A; deafness-dystonia-optic neuronopathy (DDON) syndrome; mitochondrial fission.

FIGURE 1

The female mice carrying the homologous mutation of Timm8a1-I23fs49X exhibited less gain of weight. (A) Mice at the age of 4–5 weeks [from left to right: wild-type (WT) male, MUT male, WT female, MUT female mouse]. (B) Weekly bodyweight. The Timm8a1 MUT mice were significantly smaller than the WT mice since the 3rd week (^****p < 0.0001).

FIGURE 2

Timm8a1^{I23fs49X/I23fs49X} MUT mice had hearing impairment at an early age. (A) Representative auditory brainstem responses (ABRs) (to 16 kHz pure-tone stimuli) of wild-type (WT) mice at the age of 4–5 weeks. Five positive peaks of the waveforms are shown, and wave two was labeled as PII. (B) Representative ABRs (to 16 kHz pure-tone stimuli) of MUT mice at the age of 4–5 weeks. (C) Thresholds for ABRs evoked by clicks and 8–32 kHz pure-tone pips are shown for the WT mice and the MUT mice, suggesting that the MUT mice had elevated threshold of ABRs (WT, n = 11; MUT, n = 7, *p < 0.05). Error bars represent mean ± SD.

FIGURE 3

Timm8a1^{I23fs49X/I23fs49X} MUT mice presented with mildly impaired coordinative ability and impaired maximal motor performance. (A,B) Balance beam test. The MUT mice took a similar time to cross the beam compared to the wild-type (WT) mice (WT = 8.26 ± 3.63, n = 10, MUT = 7.43 ± 2.15, n = 9, P > 0.05), while the MUT mice slipped more frequently on the balance beam than the WT mice (WT = 0.73 ± 0.44, n = 10; MUT = 1.52 ± 0.50, n = 9, ^**P < 0.01). (C) Rotarod test. The MUT mice stayed less time on the rod than the WT mice (WT = 83.65 ± 21.69, n = 10; MUT = 46.02 ± 15.44, n = 9, ^***P < 0.001). Error bars represent mean ± SD.

FIGURE 4

Timm8a1^{I23fs49X/I23fs49X} MUT mice had impaired learning and anxiety-like behavior at 20 weeks of age. (A) Traveling trace in the Morris Water Maze test. (B) From day 1 to day 4 of training, all mice showed a gradual decline in the latency to find the hidden platform over time. (C) On the probe test day, there was no difference in the spent time in the goal quadrant between the two groups (WT = 27.83 ± 11.95, n = 10; MUT = 20.34 ± 9.04, n = 9, p > 0.05). (D) On the probe test day, the MUT mice crossed less frequently in the platform area than the wild-type (WT) mice (WT = 3.00 ± 1.25, n = 10; MUT = 1.67 ± 1.22, n = 9, *p < 0.05). (E) Trace of the WT and MUT mice in the elevated plus maze test for 5 min. (F) Elevated plus maze test. The MUT mice spent less time in the open arm than the WT mice (WT = 25.93 ± 6.11, n = 9; MUT = 16.32 ± 8.87, n = 9, *p < 0.05). (G) Elevated plus maze test. The MUT mice visited the open arm less frequently than the WT mice (WT = 19.33 ± 6.02, n = 9; MUT = 12.00 ± 7.33, n = 9, *p < 0.05). Error bars represent mean ± SD.

FIGURE 5

A reduction in the mitochondrial size in the hippocampus of the Timm8a1^{I23fs49X/I23fs49X} MUT mice, correlated to upregulation of mitochondria fission protein Mtp18. (A) Histological immunostaining with an anti-NeuN antibody for neural cells in the hippocampus. (B) Electron microscopy (EM) images of the hippocampus. (C) There was no difference in the mitochondrial numbers between the two genotypes (WT = 103.20 ± 29.38, n = 5; MUT = 105.00 ± 55.22, n = 5, p > 0.05). (D) The aspect ratio was significantly decreased in the MUT mice (WT = 2.73 ± 0.36, n = 5; MUT = 1.96 ± 0.17, n = 5, ^**p < 0.01). (E) The perimeter in the MUT mice was significantly shortened (WT = 1,780.17 ± 299.25, n = 5; MUT = 1,387.43 ± 219.27, n = 5, *p < 0.05). (F) There was a tendency for less area in the MUT mice (WT = 157,451.33 ± 426,57.51, n = 5; MUT = 122,523.46 ± 423,66.67, n = 5, p > 0.05). (G) The form factor in the MUT mice was greatly reduced (WT = 1.71 ± 0.11, n = 5; MUT = 1.38 ± 0.07, n = 5, ^**p < 0.01). (H) Western blotting for the wild-type (WT) and MUT mice at 8 weeks old. (I) Mtp18/Actin is upregulated in MUT mice (WT = 0.25 ± 0.05, n = 4; MUT = 0.38 ± 0.05, n = 4, *p < 0.05). (J) There was no difference in dynamin-related protein 1 (Drp1)/Actin between the WT and MUT mice (WT = 1.08 ± 0.19, n = 4; MUT = 1.01 ± 0.09, n = 4, p > 0.05). Error bars represent mean ± SD.

FIGURE 6

Homologous mutation in the Timm8a1 gene led to a deficit in female reproductive organs. (A) The uterus and ovaries of MUT mice were morphologically smaller than that of the wild-type (WT) control at 24 weeks of age. (B) A comparison of the percentage of ovary weight to bodyweight between the WT and MUT mice (WT = 0.27 ± 0.03, n = 3; MUT = 0.05 ± 0.01, n = 3, ^***p < 0.001). (C) H&E staining of the morphology of the ovary. The preantral follicles (PreAF), antral follicles (AF), and preovulatory follicles (POF) were marked in the figures. (D) A comparison of the litter size between the WT and the MUT mice. The Mann–Whitney test was applied to analyze the difference, suggesting a decrease in fertility and litter size in MUT mice compared to WT mice (n = 6, *p < 0.05).