Loss of auditory function in transgenic Mpv17-deficient mice

ELSEVIER

Hearing Research 114 (1997) 259-.263

Loss of auditory function in transgenic Mpvl7-deficient mice Marcus Miiller a,,, Jean W.T. Smolders a, Angela M. Meyer zum Gottesberge l,, Alexander Reuter 1,d, Ralf M. Zwacka 2.,:, Hans Weiher a,d, Rainer Klinke a ~ Klinikum der J. Hd Goethe-Universitdt, Physiologisches Institut II1, Theodor-Stern-Kui 7, 60590 Franl@*rt am Main, Germany i, Universitiit Diisseldorf Forschungslabor der HNO-Klinik, Moorenstr. 5, 40255 Diisseldotf Germany ~' University o/' Pennsylvania, Institute ./br Human Gene Therapy, Stellu-Chanee-Laboratory, 422 Curie Blvd, Phihulelphia, PA 19104, USA d Forschungszentrum Karl,s'ruhe, Institut fiir Genetik, Bau 305. 76021 Karlsruhe, Germany Received 5 May 1997; revised 11 September 1997; accepted 19 September 1997

Abstract

The transgenic mouse strain Mpvl7 develops severe morphological degeneration of the inner ear and nephrotic syndrome at a young age (Meyer zum Gottesberge et al., 1996; Weiher et al., 1990). The audiograms (1 32 kHz) of Mpvl7-negative mice were determined from auditory brain stem responses in young (2 months) and old (7 months) animals. Audiograms of age-matched wildtype mice with the same genetic background, but wild-type at the Mpvl7 locus, were also determined. Furthermore, young Mpvl 7negative mice that carried a human Mpvl7 homologue gene were studied. NMRI mice served as a reference for normal hearing. Mpvl 7megative mice suffer from severe sensorineural hearing loss as early as 2 months after birth. In the old Mpvl7-negative mice no responses could be elicited at all. The 2 month old wild-type mice had normal audiograms, at 7 months only high threshold responses were seen. The poor audiograms of the Mpvl7-negative mice are assumed to be the functional correlate of the morphological degeneration of the cochlea described earlier (Meyer zum Gottesberge et al., 1996). The finding that 2 out of 4 Mpvl7-negative mice with the human Mpvl7 gene had normal audiograms, shows that the gene inactivation can be functionally compensated by the human Mpvl7 gene product. © 1997 Elsevier Science B.V.

Key words'." Mpvl7; Hearing; Audiogram; Degeneration; Glomerulosclerosis; Alport's syndrome

I. Introduction

Transgenic mice are useful as models to study deficiencies or developmental alterations in specific organs or functions. The transgenic mouse strain M p v l 7 carries a retroviral insert in its genome that inactivates the M p v l 7 gene. Y o u n g M p v l 7 - n e g a t i v e mouse m u t a n t s * Corresponding amhor. Tel.: +49 (69) 6301 6980; Fax : +49 (69) 6301 6987; E-mail: [email protected] i Present address: Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. 2 Present address: University of Edinburgh, MRC Unit Human Genetics, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, United Kingdom. a Present address: Institut fiir Diabetesforschung, K61ner Platz 1, 80804 MOnchen, Germany.

0378-5955/97/$17.00 © 1997 Elsevier Science B.V, All rights reserved PI1S0378-5955(97)00175-5

develop glomerulosclerosis and nephrotic syndrome. The nephrotic s y n d r o m e is similar to that seen in humans (Weiher et al., 1990). The discovery that inner ear pathologies also occur due to the inactivation o f the M p v l 7 gene (Meyer zum Gottesberge et al., 1996) triggered interest in the M p v l 7 mouse strain for auditory research, in these animals the stria vascularis dramatically changes its morphological appearance, and degenerative changes in structures in the organ o f Corti and cochlear nerve were described (Meyer zum Gottesberge et al., 1996). In M p v l 7 - n e g a t i v e mice, the nephrotic s y n d r o m e is first characterized by a b n o r m a l serum, blood and urine parameters. Elevated cholesterol levels, an increase in blood urea nitrogen and creatinine, and reduced albumin values are followed by a decrease in red blood cell count, low hematocrit and reduced hemoglobin levels. Finally, the animals die of renal fail-

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ure. Anatomically, the pathological changes in the kidney can be characterized as focal segmental glomerulosclerosis with hyalinosis (Weiher et al., 1990). The M p v l 7 protein was localized to the peroxisomes where it plays an unknown role in the metabolism of reactive oxygen species (Zwacka et al., 1994). The human M p v l 7 gene is structurally similar to the mouse M p v l 7 gene (Karasawa et al., 1993). It has been demonstrated that the human M p v l 7 gene can functionally rescue the glomerulosclerosis phenotype in Mpvl7-negative mice (Schenkel et al., 1995). In view of the morphological changes seen in the inner ear, which suggested a hearing impairment in Mpvl7-negative mice, the purpose of the current study was to characterize the audiograms of animals at different ages, young adult ( < 3 months) and adult ( > 6 months). These audiograms were compared to wild-type animals, the background strain of the Mpvl7-negative mice. Mpv17 transgenic mice in which the glomerulosclerosis phenotype was compensated for by a human M p v l 7 transgene (Schenkel et al., 1995) were also studied. N M R I mice, which are considered as normal hearing mice, were tested to obtain a reference audiogram.

2. Materials and methods

2.1. Mouse strains

Mice were bred and kept at the facility of the Institute of Genetics at the Forschungszentrum Karlsruhe. Mpvl7-negative mice were on a CFW/Balb/C mixed background (Weiher et al., 1990). Wild-type mice have the same genetic background but are wild-type at the M p v l 7 locus. The wild-type mice were bred alongside the mutant stock. Rescue mice are mouse Mpvl7-negative mice which carry a human M p v l 7 minigene driven by a mouse metallothioneine I gene promoter either as heterozygous or as homozygous transgene (Schenkel et al., 1995). N M R I mice were purchased from Charles River Wiga. The care and use of the animals in this study were approved by the state authorities responsible. 2.2. Audiograms

Audiograms were determined from auditory brain stem responses (ABRs). Four groups of animals were tested: N M R I (n = 2) Mpvl 7-negative (n = 8), wild-type (n = 6), Mpvl7-negative with human M p v l 7 gene insert (n = 4). Both ears were tested in all animals. All measurements were made under pentobarbital anaesthesia (60 mg/kg) administered intraperitoneally together with atropine (0.5 mg/kg). ABRs were recorded differentially from subcutaneous silver wire electrodes placed

symmetrically on both sides at positions just rostral to the pinna. The use of this symmetrical electrode arrangement reduced the artifact produced by the ECG to a minimum. A third wire, which served as ground electrode, was placed at the neck of the animal. Pure tone Gaussian shaped pips (2/3 octave bandwidth) at carrier frequencies of 1, 2, 4, 8, 16 and 32 kHz were used as acoustic stimuli. Stimuli were delivered through an inversely driven B & K 4166 microphone that was placed directly on the pinna. For signal generation, recording and analysis, a DSP-processor, 16 bit A/D and D/A modules (Tucker Davis) were used, in conjunction with an IBM compatible PC. Custom-made software was used to control the experiment. The responses to tone pips were amplified by 80 dB, bandpass-filtered (0.3-3 kHz) and recorded with an A/D converter at 20 kHz sampling rate. The recording time was 10 ms before and 10 ms after stimulus onset. The responses to tone pips with normal and inverted phase were added to eliminate cochlear microphonic responses and electrical crosstalk at high sound pressure levels. At each frequency, responses were obtained at different sound pressure levels varying from 0 to 100 dB attenuation relative to the maximum output of the system in 10 dB steps. The responses to 64 tone presentations were averaged and stored for off-line analysis. Threshold at each frequency was defined (calculated from the given attenuation) as the minimum sound pressure level (SPL) necessary for visual detection of a response in the averaged signal. The sound system was calibrated by replacing the animal's ear by a 1/4 inch B & K 4135 microphone. Mean audiograms were calculated for each animal group by averaging the audiograms. Mean thresholds at each frequency of the different groups were statistically compared by means of a t-test. The significance level was 1% throughout.

3. Results

All animals tested in this study were free of any middle ear infections. The ABR thresholds are summarized in Fig. 1. The N M R I mice (aged 6 weeks) had audiograms with best thresholds close to 20 dB SPL at 8 and 16 kHz. Fig. 1A depicts the mean audiogram of young (mean age 2.1 months) and old (mean age 7.3 months) Mpvl7negative mice. In the young Mpvl7-negative mice only responses at high sound pressure levels could be obtained, the hearing loss relative to the audiograms of the N M R ! mice amounted to 75 dB at 16 kHz and decreased to 35 dB at 4 kHz. In old Mpv17-negative mice no ABRs could be elicited at all, as indicated in the figure by plotting arrows above the maximum sound pressure level of our sound system. In Fig. 1B the audiograms of young (mean age 1.7

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however, the t h r e s h o l d s are n o t different at a n y freq u e n c y tested. In the old w i l d - t y p e mice, A B R responses were elicited o n l y at s o u n d pressure levels a b o v e 80 dB SPL. N o responses c o u l d be e v o k e d below 4 k H z , where o u r s o u n d system was u n a b l e to p r o d u c e levels a b o v e 80 dB SPL. T h e h e a r i n g loss relative to the M N R I mice a m o u n t e d to 45 dB at 4 k H z a n d increased up to 70 dB at 16 kHz. Fig. 1C gives the m e a n a u d i o g r a m s o f y o u n g (mean age 1.6 m o n t h s ) a n i m a l s negative for the m u r i n e M p v l 7 gene b u t c a r r y i n g a h u m a n M p v l 7 m i n i g e n e as a transgene. T h e results in these a n i m a l s fell clearly into two g r o u p s : O n e g r o u p h a d a u d i o g r a m s as p o o r as the y o u n g M p v l 7 - n e g a t i v e mice, the o t h e r a n i m a l s exhibited a l m o s t n o r m a l a u d i o g r a m s . T h e r e were no a n i m a l s with a u d i o g r a m s between these two types. T h e thresholds in the g r o u p with n o r m a l a u d i o g r a m s were n o t significantly ( P > 0.01) different f r o m the N M R I mice a n d the y o u n g w i l d - t y p e mice. T h e r e is h o w e v e r a t e n d e n c y for s o m e l o w - f r e q u e n c y loss (at a n d b e l o w 8 k H z ) when c o m p a r e d to the N M R I mice.

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Fig. 1. Auditory thresholds as determined from the ABR measurements in the different mouse strains at different ages. In all figures SPL refers to maximum sound pressure level (broken line), NMRI refers to the reference audiograms (n=4 ears; filled squares), vertical bars depict one standard deviation. A: Mpvl7: young (n= 10 ears; filled triangles) and old Mpvl7-negative mice (n=6 ears; arrows to indicate that thresholds were above max. sound pressure level); B: WT: young (n=6 ears; open circles) and old wild-type mice (n=6 ears; filled triangles); C: Res. 1: young mice, mouse Mpvl7-negative with human Mpvl7 gene insert and poor thresholds (n=4 ears; filled triangles); Res. 2: young mice, Mpvl7-negative with human Mpvl7 gene insert and almost normal thresholds (n = 4 ears; open circles).

m o n t h s ) a n d old ( m e a n age 7.8 m o n t h s ) w i l d - t y p e mice are shown. C o m p a r e d to the N M R ! mice the y o u n g a n i m a l s s h o w e d an a p p a r e n t l y m i n o r h e a r i n g loss at a n d a b o v e 8 kHz. A t 4 k H z a n d below, t h r e s h o l d s were c o m p a r a b l e to the N M R I animals. Statistically

4. Discussion T h e goal o f o u r s t u d y was to evaluate a u d i o g r a m s in m u t a n t mice l a c k i n g M p v l 7 p r o t e i n to d e t e r m i n e w h e t h e r the p r e v i o u s l y described inner e a r changes result in f u n c t i o n a l consequences. F u r t h e r m o r e the question o f w h e t h e r the h u m a n t r a n s g e n e was able to rescue the inner ear p h e n o t y p e as well as the k i d n e y p h e n o t y p e (Schenkel et al., 1995) was also investigated. The m e t h od used in this s t u d y to o b t a i n A B R a u d i o g r a m s revealed results similar to those o f studies in o t h e r m o u s e strains. A B R t h r e s h o l d s in N M R I mice were c o m p a r a ble to those in y o u n g N H C B A mice ( W a l t o n et al.0 1995) or y o u n g C B A / J mice ( K i r s c h et al., 1993). The N M R I mice are at least 20 dB m o r e sensitive t h a n y o u n g C 3 H / H e J mice ( T r u n e et al., 1996). T o o u r k n o w l e d g e , n o t h i n g is k n o w n a b o u t the hearing o f C F W mice ( a n c e s t o r o f M p v l 7 ) . T h e h e a r i n g c a p a b i l i t i e s o f M p v l 7 - n e g a t i v e mice were e x a m i n e d for the first time in the present study. T h e results s h o w t h a t M p v l 7 - n e g a t i v e mice suffer f r o m severe s e n s o r i n e u r a l h e a r i n g loss as early as 2 m o n t h s after birth. In a n i m a l s 7 m o n t h s o f age, no A B R s c o u l d be elicited at all at the m a x i m u m s o u n d pressure level o f the s o u n d system. It is a s s u m e d t h a t these a n i m a l s are c o m p l e t e l y d e a f at all frequencies. This confirms the conclusion o f M e y e r z u m G o t t e s b e r g e et al. (1996) t h a t the o b s e r v e d s t r u c t u r a l changes seen in the cochlea are a s s o c i a t e d with a hearing defect p h e n o t y p e . These a u t h o r s o b s e r v e d that the stria vascularis is p a r t l y disi n t e g r a t e d a n d t h a t m a r g i n a l cells are missing or a p p e a r n o t to be d e v e l o p e d at all. D e g e n e r a t i o n o f the o r g a n o f Corti, including loss o f inner a n d o u t e r hair cells, a n d

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loss of spiral ganglion cells was reported. Furthermore, the morphological changes were more severe in older than in younger animals. This agrees with the findings described here. In our younger animal group at least some responses to tone pips could be elicited, whereas in the older animals no A B R responses could be recorded. The m o r p h o l o g y also revealed a gradient in the cochlear changes showing more severe lesions in the basal turns of the cochlea compared to apical turns. This suggests that hearing loss begins at higher frequencies and proceeds to lower frequencies. In fact, in young Mpvl7-negative mice (2 months old) hearing loss is most prominent at high frequencies. The hearing loss amounted to about 75 dB at 16 kHz, and to only 35 dB at 4 kHz. The stria vascularis is the main organ which generates the endocochlear potential, the driving force for the transduction process in the cochlea. Changes in the endocochlear potential (Sewell, 1984; Vossiek et al., 1991) influence nerve fibre sensitivity and activity. Degeneration of inner and outer hair cells, which was also observed, is known to be one major reason for ganglion cell degeneration (e.g. Schuknecht, 1974). At present it is unclear whether the lack of endocochlear potential might be responsible for hair cell degeneration, or whether hair cell degeneration is caused directly by the loss of function of the M p v l 7 gene. Despite a lack of detectable morphological alterations in the cochleae (Meyer zum Gottesberge et al., 1996), the wild-type strain ( C F W × Balb/C) also develops a hearing impairment which begins at two months of age and leads to severe hearing loss at seven months. This degeneration at a young age is not u n c o m m o n in inbred mice strains (e.g. Dazert et al., 1996; Keithley et al., 1992) and suggests that a degenerative inner ear disease may be present in the predecessors. The rescue of the phenotype observed in mice negative for the endogenous M p v l 7 gene but transgenic for the h u m a n M p v l 7 homologue was demonstrated here. This was, however, only found in half of these transgenic animals. Preliminary data (dot-blot analysis from two additional animals) suggest a possible hypothesis to explain the finding: H a l f of the mice are heterozygous and the other half homozygous for the h u m a n M p v l 7 transgene. Unlike the case with glomerulosclerosis (Schenkel et al., 1995), it appears that one copy of the transgene is not sufficient for a functional rescue of the hearing deficit phenotype. The data suggest that in order to compensate for the hearing phenotype the mice have to be homozygous for the transgene. The time course of the development of hearing deficits in Mpvl7-negative mice correlates well with the onset of the nephrotic syndrome. This was observed in this mutant at two months of age (Weiher et al., 1990). However, the finding that severe sensorineural hearing loss occurs when the kidneys of the animals

are still functionally intact and just begin to develop deficiencies, implies that the inner ear is much more vulnerable to the missing M p v l 7 gene product. It is also possible that the lack of M p v l 7 gene product accelerates the existing degenerative inner ear disease seen in the background strain. It is possible that full expression of both M p v l 7 alleles is necessary for proper inner ear function but that the kidney function remains normal at lower levels of gene expression. This view is supported by the findings in the rescue mice. The phenotype of the Mpvl7-negative mice is remarkably similar to Alport's syndrome, a genetic disease found in humans in which collagen IV genes are mutated (Netzer et al., 1993). The hereditary incidence of nephrotic syndrome and the occurrence of sensorineural hearing loss is termed Alport's syndrome (A1port, 1927; for review see Schuknecht, 1974; Tryggvason, 1996). Since no cases of familial glomerulosclerosis could be related to the M p v l 7 gene so far, the molecular defect is certainly different from that in Alport's syndrome. However, studying pathology in Mpvl7-negative mice m a y be useful in understanding genetically linked kidney and inner ear pathology. Both kidney and cochlear structures and functions, require a very specific ionic environment for proper function. The possible correlation between cochlear and kidney failure (e.g. Arnold, 1984; Weidauer and Arnold, 1976) might be accounted for by some c o m m o n mechanism to maintain or produce this ionic environment. The Mpvl7-deficient mice might represent a good model to study such comm o n mechanisms, both morphologically and functionally.

Acknowledgments The study was supported by the Deutsche Forschungsgemeinschaft, SFB 269, B1 and M E 890/4-1.

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