Spermine synthase deficiency leads to deafness and a profound sensitivity to alpha-difluoromethylornithine

Abstract

Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the SpmS and Phex genes, were found to be profoundly hearing impaired. This defect was due to alteration in polyamine content due to the absence of spermine synthase, the product of the SpmS gene. It was reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that ubiquitously expresses spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken beta-actin promoter. There was an almost complete loss of the endocochlear potential in the Gy mice, which parallels the hearing deficiency, and this was also reversed by the production of spermine from the spermine synthase transgene. Gy mice showed a striking toxic response to treatment with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO). Within 2-3 days of exposure to DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor function resulting in death within 5 days. This effect was due to an inability to maintain normal balance and was also prevented by the transgenic expression of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no effect on balance. The loss of balance in Gy mice treated with DFMO was due to inhibition of polyamine synthesis because it was prevented by administration of putrescine. Our results are consistent with a critical role for polyamines in regulation of Kir channels that maintain the endocochlear potential and emphasize the importance of normal spermidine:spermine ratio in the hearing and balance functions of the inner ear.

FIGURE 1.

Hearing threshold in control, Gy, SPS, and SPS/Gy mice. The SPL in dB of broadband clicks (0.1 ms) delivered to the ear are indicated on the left side of the traces. Representative normal broadband click responses from control (A), SPS (B), Gy (C), and SPS/Gy (D) mice are shown. The auditory brainstem response responses over the range of 30–100 dB for representative mice from each group are shown. No response was observed from any of the 10 Gy mice examined. E, auditory brainstem response thresholds for wild type (n = 36), SPS (n = 12), and SPS/Gy (n = 12) mice in response to broadband clicks and 3-ms pure tones of 8, 16, and 32 kHz (p = 0.5 at 8 kHz, p = 0.6 at 16 kHz, and p = 0.4 at 32 kHz). The data are means ± S.D. Gy mice showed no detectable response to any stimulus.

FIGURE 2.

DPOAE testing of control, Gy, SPS, and SPS/Gy mice. Mean distortion products for 7–8-week-old control (wild type, n = 21), Gy (n = 7), SPS (n = 7), and SPS/Gy mice were tested measuring the levels of the 2 f₁ – f₂ DPOAE over a geometric-mean frequency range from 5.6 to 48.5 kHz, using an f₂/f₁ of 1.25 and primary tone stimuli at L₁ = L₂ = 65 SPL. Clearly, Gy mice yielded no significant DPOAEs at low-to-high frequencies (5.6–45 kHz) when compared with controls, SPS, and SPS/Gy mice.

FIGURE 3.

EP in control, Gy, SPS, and SPS/Gy mice. Gy mice had a significant reduction in EP at the basal turn of the cochlea, control/wild type (WT) = 108 ± 14 mV (n = 6); SPS = 92 ± 17 mV (n = 4); Gy = 9 ± 7 mV (n = 4) and SPS/Gy = 89 ± 12 mV. EP data obtained from the apical turn were wild type = 101 ± 8 mV (n = 3); SPS = 91 ± 13 (n = 3); Gy = 6 ± 5 (n = 3), and SPS/Gy = 85 ± 14 mV (n = 3).

FIGURE 4.

Histology of Scala media of the lower apical turn of the cochlea in control (A), SPS (B), SPS/Gy (C), and Gy (D) mice. A, a cross-section of the scala media from a control cochlea shows a normal complement of spiral ganglion cells (SGC), and organ of Corti (OC) with inner hair cell (arrowhead) and three outer hair cells (double asterisk), each supported by a Deiters cell (asterisk). The lateral wall consists of the stria vascularis (StV) of expected density and thickness (black bar) and fibrocytes of the spiral ligament (SpLig). B, the cross-section of the SPS cochlea appears normal. Increased thickness (black bar) is noted in the stria vascularis. C, increased strial width (black bar) is the only aberrant feature noted in the cochlea of the SPS/Gy mouse. D, the cochlea of the Gy mouse lacks eighth nerve fibers (8^th n.) and spiral ganglion cells. The hair cells and supporting cells of the organ of Corti have been replaced by a layer of squamous epithelia cells (arrow). The fibrocytes of the spiral ligament (SpLig), and the stria vascularis (StV) appear grossly normal. SpL, spiral limbus.

FIGURE 5.

Balance index in control, Gy, SPS, and GySPS mice before and after 1.5-day treatment with DFMO. The times taken to remain on a rotating cylinder (5 rpm) were defined as the balance index as described (49) and are illustrated in the histogram. Data were obtained from DFMO pre- and post-treated mice. Seven animals from each genotype were tested. The mean of 10 trials for each animal is reported. The asterisk indicates significant differences (p < 0.01) between pre- and post-treated animals. To eliminate fatigue factor, each trial was separated by a 10-min rest period. No reward was given to the animals during or after the test. Unshaded bars show results after DFMO treatment. WT, wild type.

FIGURE 6.

Effect of l-ornithine and DFMO on body weight of Gy mice. Three Gy mice (4–6 weeks in age) were exposed to l-ornithine by placing 1% in the drinking water for 5 days, and the drinking water was then replaced by water with no additions for 5 days and then changed to water containing 1% DFMO. The body weight of each mouse was measured as shown. No behavioral changes were seen until the DFMO administration, when all mice developed the inability to maintain posture described under “Results.”