From the Departments of Plasma membrane Ca2+-ATPase
isoform 2 (PMCA2) exhibits a highly restricted tissue distribution,
suggesting that it serves more specialized physiological functions than
some of the other isoforms. A unique role in hearing is indicated by
the high levels of PMCA2 expression in cochlear outer hair cells and
spiral ganglion cells. To analyze the physiological role of PMCA2 we
used gene targeting to produce PMCA2-deficient mice. Breeding of
heterozygous mice yielded live homozygous mutant offspring. PMCA2-null
mice grow more slowly than heterozygous and wild-type mice and exhibit
an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice
revealed that null mutants had slightly increased numbers of Purkinje
neurons (in which PMCA2 is highly expressed), a decreased thickness of
the molecular layer, an absence of otoconia in the vestibular system,
and a range of abnormalities of the organ of Corti. Analysis of
auditory evoked brainstem responses revealed that homozygous mutants
were deaf and that heterozygous mice had a significant hearing loss.
These data demonstrate that PMCA2 is required for both balance and
hearing and suggest that it may be a major source of the calcium used
in the formation and maintenance of otoconia.
Calmodulin-dependent plasma membrane
Ca2+-ATPases
(PMCAs)1 are highly regulated
enzymes that maintain the appropriate concentrations of intracellular
free Ca2+ by extruding Ca2+ from the cell (1,
2). There are four mammalian PMCA isoforms (PMCA1-4), each encoded by
a distinct gene (3-8), and additional diversity is generated by
alternative splicing of exons encoding the regulatory domains (7,
9-11). Variants of PMCA1 and PMCA4 are expressed in many different
tissues and cell types, whereas variants of PMCA2 and PMCA3 exhibit a
highly restricted distribution (5, 8, 12). This suggests that specific
isoforms and splice variants serve different physiological functions.
However, despite extensive information about PMCA structural diversity,
expression patterns, and biochemical and regulatory characteristics,
little is known about the functions of individual isoforms in
vivo.
Its unique biochemical characteristics (13, 14) and tissue specificity
(5, 10-12) suggest that PMCA2 might serve specialized physiological
functions. In situ hybridization studies revealed that
expression of PMCA2 is particularly high in Purkinje neurons of the
cerebellum (15) and in the spiral ganglion nerves of the inner ear and
outer hair cells of the cochlea (16). The observation that PMCA2 is the
predominant isoform in outer hair cells (16) suggests that it might be
the isoform that is expressed at high levels in stereocilia (17), which
comprise hair bundles, the sensory organelles that mediate
mechanoelectrical transduction by hair cells of both the vestibular and
auditory systems (18, 19). A recent study demonstrated that PMCA
activity in the stereocilia of vestibular hair cells regulates hair
bundle Ca2+ concentrations and indicated that it might also
control the Ca2+ concentrations in the endolymph
immediately surrounding the hair bundles (20). The results of our
analyses of the PMCA2-deficient mouse, described in the current study,
demonstrate that PMCA2 plays critical roles in both the vestibular and
auditory systems.
Gene Targeting and Hybridization Analyses--
The targeting
construct was prepared using fragments of the mouse
Pmca22 gene and
the vector, pMJKO, which was described previously (21). A 2.0-kb
BspHI fragment beginning in intron 17 and terminating within
codon 958 in exon 19 was inserted into a cloning site between the neo
gene and the herpes simplex virus thymidine kinase gene in the vector.
A 2.3-kb BspHI-NotI fragment beginning within
codon 958 and terminating in exon 21 was inserted into another cloning site 5' to the promoter for the neo gene. Techniques for targeting of
ES cells and generation of mutant mice were the same as those used
previously (21). Southern blot analysis of ES cell and tail DNAs was
performed using a 5' probe containing parts of exon 17 and intron 17 and a 3' probe containing parts of intron 20 and exon 21. Northern blot
analysis was carried out as described previously (21) using a cDNA
probe spanning nucleotides 22-519 of the rat PMCA2 5'-untranslated
sequence (3) and total RNA from brains of 6-8-week-old mice.
Histology and Morphometry--
Cerebella and intact inner ears
from 5-6-week-old mice of all three genotypes were removed after
perfusion fixation with a phosphate-buffered saline solution containing
paraformaldehyde and gluteraldehyde. Temporal bones were decalcified in
EDTA for 1 week. The specimens were post-fixed in buffered 1% osmium
tetroxide for 1-2 h, dehydrated in a gradient of ethanol washes and
propylene oxide, and embedded in Spurr's resin. Serial 1-2-micron
sections perpendicular to the folia of the cerebellum
(n = 9 Pmca2+/+, 11 Pmca2+/ Molecular Genetics,
Cell Biology and Anatomy, and
** Environmental Health, University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267, the ¶ Department of Biological Sciences,
University of Cincinnati, Cincinnati, Ohio 45221, and the
§ House Ear Institute, Los Angeles, California 90057
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, and 8 Pmca2
/
mice) or parallel to the modiolus of
the inner ear (n = 8 Pmca2+/+, 8 Pmca2+/
, and 10 Pmca2
/
mice) were cut and stained with
toluidine blue.
Auditory Brainstem Responses-- Mice were anesthetized with avertin, and ABR measurements were performed within a sound-attenuated chamber as described previously (22). Three electrodes, one under each ear and one at the top of the skull, were inserted subcutaneously. The mice were placed between two high frequency transducers, and tubing was oriented to direct sound stimuli into the ear canals. The ABR was computer-averaged (time-locked with onset of 128-1024 stimuli, at 20/s) out of the continuous electroencephalographic activity, and the threshold of hearing was determined by observing the lowest intensity of sound required to elicit a characteristic waveform.
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RESULTS |
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Generation of Mutant Mice--
The gene encoding PMCA2
(Atp2b2) was disrupted in ES cells using a targeting
construct (Fig. 1A) in which
the neo gene was inserted into exon 19, which encodes transmembrane
domains essential for transport activity. Chimeric male mice were
prepared using these ES cells and bred with wild-type Black Swiss mice.
Germline transmission of the mutant allele was achieved, and breeding
of Pmca2+/ mice yielded live offspring of all
three genotypes (Fig. 1B) in a 1:2:1 Mendelian ratio (74 +/+, 151 +/
, 87
/
). Northern blot analysis demonstrated that the
mutation caused a reduction of PMCA2 mRNA expression in brain of
Pmca2+/
mice and eliminated expression in
Pmca2
/
mice (Fig. 1C).
|
Gross Phenotype--
Homozygous mutant mice grew to adulthood
(with the oldest currently at 8 months of age) with no excess
mortality, but they exhibited severe ataxia that was clearly apparent
by 12 days of age and had reduced body weight (~20-30% less than
Pmca2+/ and Pmca2+/+
mice). Pmca2+/
mice did not display these
outward manifestations of an abnormal phenotype. As shown in
panels A-C of Fig. 2, young
Pmca2
/
mice had great difficulty maintaining
their balance when walking or standing and frequently fell onto their
sides. The mice often rolled onto their backs and would flail their
hind legs and tail while attempting to right themselves. By 5 months of
age, the Pmca2
/
mice improved in their
ability to maintain an upright position, and many of them tended to
alternately hyperextend (in the manner shown in Fig. 2B) and
clench their rear legs and feet together (Fig. 2D). In
addition to the apparent balance deficit,
Pmca2
/
mice showed no response to a hand
clap, indicating that their hearing was impaired.
|
Histological and Morphometric Analyses of Cerebellum--
Because
PMCA2 is expressed at high levels in Purkinje neurons (15), we
evaluated the morphology of the cerebellum. Heterozygous mice were not
significantly different from wild-type mice in any of the measured
parameters. In null mutants, the numerical density of Purkinje cells
was increased (2.4 ± 0.0 and 1.9 ± 0.1 cells/100 microns
for Pmca2/
and
Pmca2+/+ mice, respectively; p < 0.02), and the numerical density of granule cells was reduced
(6.4 ± 0.24 and 7.7 ± 0.14 cells/100 microns for
Pmca2
/
and Pmca2+/+
mice, respectively; p < 0.01). There was no
significant difference in the staining intensity of Purkinje cells, but
the percentage of granule cells that were densely stained (pyknotic)
was increased (3.9 ± 1.5% for Pmca2
/
and 0.28 ± 0.16% for Pmca2+/+,
p < 0.01). Finally, the molecular layer was thinner in
Pmca2
/
mice (171 ± 10 microns for
Pmca2
/
and 206 ± 11 microns for
Pmca2+/+, p < 0.05).
Histology of the Inner Ear--
Light microscopic analysis of
sections of the vestibular system from mice of all three genotypes
revealed no apparent histopathology of the semicircular canals or
cristae ampulares. Hair cells, support cells, and innervation of the
sensory epithelium in the utricle and saccule of both wild-type and
mutant mice appeared normal, and the otolithic membrane was present
above the macula in each chamber. Whereas otoconia were numerous and
clearly visible in the vestibular organs of
Pmca2+/+ mice (as shown in the saccule, Fig.
3A) and were also observed in
the vestibule of Pmca2+/ mice (data not
shown), these calcium carbonate crystals were missing in both the
saccule (Fig. 3B) and utricle (data not shown) of
Pmca2
/
mice.
|
|
Auditory Brainstem Responses Demonstrate That
Pmca2/
Mice Are Deaf--
To test the hypothesis that
the Pmca2 mutation might cause a hearing deficit, auditory
brainstem responses were analyzed to determine the sound pressure level
at which the characteristic ABR waveform could be detected. The sound
stimuli used were a broadband click and pure tones of 8, 16, and 32 kHz. Pmca2+/+ mice yielded the characteristic
ABR wave form, beginning at sound pressure levels of ~30-45 db. In
the example shown in Fig. 5A, all five ABR peaks are evident for the 50, 60, and 70 db stimulus intensities, and the relatively small first and second peaks are present for the detected threshold at 40 db. In contrast, none of the
Pmca2
/
mice exhibited a detectable waveform
even at 99 db (Fig. 5A). As shown in Fig. 5B,
Pmca2+/
mice exhibited markedly elevated ABR
thresholds (~70-80 db) for each of the four sound stimuli, when
compared with the thresholds (~30-45 db) for wild-type mice.
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DISCUSSION |
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The objectives of this study were to develop a Pmca2-deficient mouse and to analyze its phenotype to better understand the physiological functions of this isoform in vivo. The absence of detectable PMCA2 mRNA in homozygous mutants confirmed that our targeting strategy had produced a null mutation. Homozygous mutants were born in the normal Mendelian ratio and exhibited no excess mortality, demonstrating that PMCA2 is not essential for embryonic development or survival after birth. Severe ataxia, however, was clearly apparent before 2 weeks of age, and additional studies revealed defects of both the vestibular and auditory systems.
Because in situ hybridization studies have shown that PMCA2
is expressed at high levels in Purkinje neurons (15), we considered the
possibility that the ataxia was because of a cerebellar defect. Morphological alterations in the Pmca2/
cerebellum included a decrease in the number of granule cells, a slight
increase in the number of Purkinje cells, and a decreased thickness of
the molecular layer, which suggests the possibility of decreased
arborizations of the Purkinje cell dendritic processes. Although it is
conceivable that these changes contribute to the phenotype, they seem
minor compared with those seen in the pcd (Purkinje cell degeneration) mutant
mouse, which loses most of its Purkinje cells yet shows only moderate
ataxia (23). This led us to consider the possibility of defects in the
vestibular system, which senses equilibrium and balance (18).
The vestibular system consists of the semicircular canals, which detect
changes in angular acceleration, and the utricle and saccule, which
detect changes in linear acceleration and head position with respect to
gravity. Histological abnormalities were not observed in the
semicircular canals of Pmca2/
mice, and the
crista ampullaris, with its sensory hair cells and gelatinous cupula
within the ampulla of each duct, appeared normal. Similarly, we
observed no abnormalities of the maculae of the saccule and utricle,
and in each chamber the gelatinous otolithic membrane that overlies the
neuroepithelium was intact. Pmca2-null mutants, however,
lacked otoconia in both the saccule and utricle. Because these calcium
carbonate crystals are very dense relative to the endolymph and
otolithic membrane, their mass is readily shifted in response to
gravity or linear acceleration, thereby shifting the otolithic membrane
and stimulating the hair cells. The fact that null mutants exhibited
difficulties in maintaining their balance even when standing still,
which would not involve the semicircular canals, is consistent with a
deficit in the saccule and utricle. Thus, the lack of otoconia provides
a striking histopathological correlate of the defect in balance.
Recent evidence indicates that otoconia grow by accretion of calcium
carbonate prior to and shortly after birth (24), although the mechanism
by which they are formed and the source of the calcium is not known.
There is no information about the expression of PMCA2 in the vestibular
system, but a recent study has shown that it is expressed in multiple
cell types bathed by the endolymph of the cochlear duct (16), which is
continuous with the membranous labyrinth of the vestibular system. The
absence of otoconia in Pmca2/
mice raises
the interesting possibility that these calcium carbonate crystals are
formed and/or maintained, at least in part, by Ca2+
extruded into the endolymph by PMCA2.
The most striking observations were the hearing deficits and
histopathology of the organ of Corti in both
Pmca2/
and Pmca2+/
mice. ABR measurements demonstrated that
Pmca2
/
mice are profoundly deaf and that
Pmca2+/
mice exhibit a severe hearing loss.
The hearing loss in heterozygous mutants is of considerable interest
because it shows that a null mutation in the Pmca2 gene can
cause an autosomal dominant hearing loss. The observed histopathology
is consistent with the possibility that PMCA2 plays a role in the
development of the organ of Corti and spiral ganglion neurons; however,
there were regions in which these structures appeared relatively
normal. Thus, an alternative possibility is that the organ of Corti and
spiral ganglion neurons develop normally and then degenerate with age,
as seen in the deafness mouse (25). The expression of PMCA2
in outer hair cells (16) suggests that it is the isoform that is
present at high levels in the hair bundle (17, 26) and might therefore
play an important role in sensory transduction. PMCA activity in the hair bundle has been shown to regulate Ca2+ concentrations
within the stereocilia and may also generate a Ca2+
gradient near the stereocilia that might influence the sensitivity of
the hair cell (20). Additional studies will be needed to determine the
time course and mechanisms underlying the histopathology of the organ
of Corti and whether PMCA2 plays a direct role in mechanoelectrical
transduction by hair cells.
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ACKNOWLEDGEMENTS |
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We thank Allen F. Ryan for sharing the results of work on the localization of PMCA2 mRNA prior to publication, Jeannette Greeb for assistance in genotype analysis, Stacey Andringa, Phillip Sanford, and Nancy Paradies for assistance in preparing samples for histology, and Paul Esmahan for assistance in preparing the illustrations.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK50594, HL41496, ES06096, and TG HL07382 and a grant from the National Organization for Hearing Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should addressed: Dept. of Molecular
Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Bethesda Ave., ML 524, Cincinnati, OH
45267-0524.
1 The abbreviations used are: PMCA, plasma membrane Ca2+-ATPase (the number following PMCA refers to the specific isoform); neo, neomycin resistance; kb, kilobase(s); ES, embryonic stem; ABR, auditory brainstem responses.
2
Pmca2+/+,
Pmca2+/, and Pmca2
/
are wild-type, heterozygous, and homozygous mutant mice, respectively
(by convention, when referring to the mouse gene, only the first letter
is in uppercase letters).
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REFERENCES |
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