Loss of Murine Na+/myo-Inositol Cotransporter Leads to Brain myo-Inositol Depletion and Central Apnea
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m213176200
ISSN1083-351X
AutoresGerard T. Berry, Shuang Wu, Roberto Buccafusca, Jun Ren, Linda W. Gonzales, Philip L. Ballard, Jeffrey A. Golden, Martin J. Stevens, John J. Greer,
Tópico(s)Infant Nutrition and Health
Resumomyo-Inositol (Ins) and its polyphosphoinositide derivatives that are important in membrane signaling have long been held to play a special role in brain metabolism. As polyphosphoinositides turn over rapidly and are exceptionally abundant in nervous tissue, high Ins levels in the range of 2–15 mm that have been observed in brain may be necessary to maintain the rates of phosphoinositide synthesis in diverse membrane locations within neurons. Cellular concentration gradients of this magnitude indicate a dependence on active Ins transport, especially at the time of growth and differentiation. The Na+/myo-inositol cotransporter (SMIT1 or SLC5A3) gene is highly expressed prenatally in the central nervous system and placenta. To gain more insight into brain Ins metabolism, while ascertaining the importance of SMIT1 as a transporter, we generated mice with a homozygous targeted deletion of this gene. Newborn SMIT1(−/−) animals have no evidence of SMIT1 mRNA, a 92% reduction in the level of brain Ins, an 84% reduction in whole body Ins, and expire shortly after birth due to hypoventilation. Gross pathologic and light microscopic examinations of each organ, as well as the placenta, of embryonic day 18.5 fetuses at near term gestation were normal. Based on [3H]acetate incorporation into phospholipids of lung tissue explants, immunostaining of lung tissue for surfactant protein A, B, and C, and electron microscopic examination of alveolar cells, there was no evidence of abnormal pulmonary surfactant production by type 2 pneumocytes in lung. Although no histologic lesions were detected in the nervous system, electrophysiological studies of the brainstem pre-Bötzinger respiratory control center demonstrated an abnormal rhythm discharge with periods of central apnea. The cause of death can be explained by the regulatory defect in brainstem control of ventilation. This model demonstrates the critical importance ofSMIT1 in the developing nervous system. The high affinity SMIT1 transporter is responsible for the Ins concentration gradient in the murine fetal-placental unit. myo-Inositol (Ins) and its polyphosphoinositide derivatives that are important in membrane signaling have long been held to play a special role in brain metabolism. As polyphosphoinositides turn over rapidly and are exceptionally abundant in nervous tissue, high Ins levels in the range of 2–15 mm that have been observed in brain may be necessary to maintain the rates of phosphoinositide synthesis in diverse membrane locations within neurons. Cellular concentration gradients of this magnitude indicate a dependence on active Ins transport, especially at the time of growth and differentiation. The Na+/myo-inositol cotransporter (SMIT1 or SLC5A3) gene is highly expressed prenatally in the central nervous system and placenta. To gain more insight into brain Ins metabolism, while ascertaining the importance of SMIT1 as a transporter, we generated mice with a homozygous targeted deletion of this gene. Newborn SMIT1(−/−) animals have no evidence of SMIT1 mRNA, a 92% reduction in the level of brain Ins, an 84% reduction in whole body Ins, and expire shortly after birth due to hypoventilation. Gross pathologic and light microscopic examinations of each organ, as well as the placenta, of embryonic day 18.5 fetuses at near term gestation were normal. Based on [3H]acetate incorporation into phospholipids of lung tissue explants, immunostaining of lung tissue for surfactant protein A, B, and C, and electron microscopic examination of alveolar cells, there was no evidence of abnormal pulmonary surfactant production by type 2 pneumocytes in lung. Although no histologic lesions were detected in the nervous system, electrophysiological studies of the brainstem pre-Bötzinger respiratory control center demonstrated an abnormal rhythm discharge with periods of central apnea. The cause of death can be explained by the regulatory defect in brainstem control of ventilation. This model demonstrates the critical importance ofSMIT1 in the developing nervous system. The high affinity SMIT1 transporter is responsible for the Ins concentration gradient in the murine fetal-placental unit. myo-inositol phosphatidylinositol Na+/myo-inositol cotransporter1 gas chromatography/mass spectrometry surfactant protein phospholipid phosphate-buffered saline trimethysilylation phosphoglycerokinase N,O-bis(trimethylsilyl)trifluoroacetamide trimethylchlorosilane embryonic day myo-Inositol (Ins)1 and its polyphosphoinositide derivatives that are important in membrane signaling have long been held to play a special role in brain metabolism (1Dawson R.M. 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The murine 129svJ genomic library was from Strategene (La Jolla, CA). The restriction endonucleases were from Promega (Madison, WI). The PGK-neomycin cassette was a gift from Dr. Nancy Cooke. The HSV-tk-Dt α subunit cassette was a gift from Dr. Barbara Knowles. The myo-inositol andN,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS) mixture were from Sigma. The hexadeuterated myo-inositol ([2d6]Ins) was from CDN Isotopes (Quebec, Canada). The Waymouth's MB 752/1 medium and phosphate-buffered saline (PBS) were purchased from Invitrogen. The paraformaldehyde and glutaraldehyde were from Electron Microscopy Sciences (Ft. Washington, PA). The Triton X-100 was from Roche Molecular Biochemicals. The [3H]acetate (100 mCi/mMol),myo-[2-3H]inositol (22.3 Ci/mmol), and [32P]dCTP (3000 Ci/mmol) were from PerkinElmer Life Sciences. The halothane was from Abbott Laboratories, Inc. The sodium borohydride, Superfrost glass slides and Tissue Freezing Medium were from Fisher. All other chemicals were from Sigma. Previously, we cloned a genomic fragment that contained the intron-free coding region of the mouse SMIT1 homologue (33McVeigh K.E. Mallee J.J. Lucente A. Barnoski B.L. Wu S. Berry G.T. Cytogenet. Cell Genet. 2000; 88: 153-158Crossref PubMed Google Scholar) from a 129svJ genomic library. The fragment is ∼15 kb and exists in a λ bacteriophage vector. From this clone, an 11.0-kbXbaI fragment was subcloned and used to construct a targeting vector for homologous recombination by positive-negative selection. A 1.6-kb EcoRI-NcoI fragment within the SMIT1 coding region was replaced with a 1.9-kb PGK-neomycin cassette (see Fig. 1). A HSV-tk-Dt α subunit cassette was ligated to the 3′-end of the vector to allow negative selection of the non-targeting events. Embryonic stem cells (129svJ) were electroporated, and G418-resistant clones were isolated in the laboratories of GenomeSystems, St. Louis, MO. In the laboratory of Dr. Gerard T. Berry, a 1-kb SacI fragment 5′to the homology contained in the targeting vector was used as a probe in Southern blot analyses to identify homologous recombination events. HindIII digestion of the wild-type locus generates a 6.5-kb fragment detectable by probe 1, whereas the correctly targeted locus yields a 10-kb fragment (see Fig. 1). Twelve correctly targeted clones were identified among 192 resistant clones, yielding a targeting frequency of 1 in 16. In the Transgenic Animal Facility of the University of Pennsylvania, one embryonic stem clone was selected for blastocyst injection; of eight chimeric mice obtained, four transmitted embryonic stem cell DNA through the germ line and generated heterozygous offspring. Germline transmission was demonstrated by Southern blot analysis of tail DNA. The content of SMIT1 transcript in adult, fetal, and placental tissues was determined by Northern blot analyses. To maximize the sensitivity and specifically of Ins detection, we employed GC/MS using hexadeuterated Ins ([2d6]Ins) in an isotope dilution analysis. We employed trimethysilylation (TMS) and an Hewlett-Packard 5890/5972 analyzer equipped with electron ionization in the selected ion monitoring mode (3Godfrey D.A. Hallcher L.M. Laird M.H. Matschinsky F.M. Sherman W.R. J. Neurochem. 1982; 38: 939-947Crossref PubMed Scopus (16) Google Scholar). The selected ion monitoring fragments of interest are 217 for TMS-Ins and 220 for TMS-[2d6]Ins. We generated a linear standard curve using 1000 pmol of [2d6]Ins per derivatization vial, with Ins varying from 200 to 1800 pmol. The final derivatization volume containing BSTFA/TMCS with standards was 300 μl, and 1 μl was used for each GC/MS injection. Based upon our results, this method will permit picomolar amounts of Ins to be reliably assayed. The isotope dilution analysis with [2d6]Ins was used to measure Ins levels in whole embryonic day (E) 10.5, E14.5, and E18.5 fetuses from sets of litters and in amniotic fluid samples from one of the E16.5 sets. The fetuses were quickly frozen in liquid nitrogen after removal from the uterine sacs. The yolk sac DNA was used for genotyping. A 5-μl sample of amniotic fluid was frozen and lyophilized. The whole fetus was homogenized following addition of [2d6]Ins. Aliquots were taken for protein assay. Following lyophilization of fetal extracts, the TMS-derivatized samples were analyzed by GC-MS. On day 18.5 of gestation, the uterus with fetuses intact was removed and placed in sterile PBS on ice, and each fetal lung was dissected out. Of the three lobes on the right side, one was fixed for EM, one was fixed for immunostaining, and the third (largest) was cultured with myo-[2-3H]inositol for labeling of PtdIns. Of the two lobes on the left side, one was frozen (−70 °C) for later use, and one (larger) was cultured for [3H]acetate incorporation into all phospholipids. Lobes were placed into individual wells of 24-well culture dishes with the precursor (250 μl of Waymouth's media with either [3H]acetate (20 μCi/ml, 1 mm total acetate) or myo-[2-3H]inositol (10 μCi/ml, 7 μm Ins) and rocked for 5 h at three cycles/min in a 37 °C humidified incubator. Concentration of acetate was high (1 mm) to ensure rapid equilibration of the endogenous pools and eliminate pool effects. After incubation, the tissues were harvested into cold PBS, and then sonicated in saline and assayed for protein. To determine distribution of incorporation into newly synthesized phospholipids, total lipid extracts were separated by thin-layer chromatography (TLC), and the amount of tritium label in each PL was determined as described previously (34Gonzales L.W. Ballard P.L. Ertsey R. Williams M.C. J. Clin. Endocrinol. Metab. 1986; 62: 678-691Crossref PubMed Scopus (160) Google Scholar). To prepare frozen tissue sections for immunofluorescence, lobes were fixed in 1% paraformaldehyde in PBS overnight at 4 °C, washed with PBS for 5 min, and embedded in Tissue Freezing Medium (Fisher). Cut sections were washed (3 min) with sodium borohydride (0.1% in PBS) to reduce autofluorescence and rinsed twice with PBS (5 min). Sections were incubated (30 min, 25 °C) in PBS containing 0.3% Triton X-100 + 5% bovine serum albumin + 10% normal goat serum to block nonspecific binding and permeabilize cells followed by a 5-min wash with PBS + Triton X-100. Coverslips were incubated with primary antibodies overnight at 4 °C. Antibodies used were: rabbit anti-human SP-A (polyclonal), anti-bovine SP-B (polyclonal prepared against SP-B extracted from bovine surfactant), and anti-rat SP-C (polyclonal which recognizes precursor forms) as described previously (35Beers M.F. Solarin K.O. Guttentag S.H. Rosenbloom J. Kormilli A. Gonzales L.W. Ballard P.L. Am. J. Physiol. 1998; 275: L950-L960PubMed Google Scholar). To remove excess antibody, slides were incubated with PBS + Triton X-100 for 5 min. Primary antibodies were detected by addition of secondary antibody (goat anti-rabbit IgG conjugated to Cy3, 1:200) for 1 h at 25 °C. Excess secondary antibody was removed by 2-min washes (twice each) with PBS + 0.3% Triton X-100, and then with PBS + 0.075% Triton X-100, and finally with PBS. Coverslips were air-dried and mounted with Mowiol (Calbiochem). Fluorescence was examined with an Olympus 1X70 microscope and Metamorph imaging system. For electron microscopy, lobes were fixed in 2.5% glutaraldehyde, 0.1m sodium cacodylate (pH 7.2) for 3 h at 4 °C, washed, and postfixed with 1% osmium tetroxide. The tissue was embedded in epoxy resin, and ultrathin sections were contrasted with uranyl acetate and lead citrate and examined in a JEOL CX100II transmission electron microscope operated at 80 kV. Cells from two lungs of each group were examined (four sections/sample). The screening of respiratory motor pattern was double-blinded as neither the genotype of the fetus was known at the time of this recording nor were the results of the electrophysiological testing known by the individual performing the Southern blot. Fetuses (E18.5) were delivered from timed-pregnant mice anesthetized with halothane (1.25–1.5% delivered in 95% O2 and 5% CO2) and maintained at 37 °C by radiant heat. The timing of pregnancies of dams was determined from the appearance of sperm plugs in the breeding cages. Embryos were immediately decerebrated, and the brainstem/spinal cord with the ribcage and diaphragm muscles attached was dissected following procedures similar to those established for perinatal rats (36Smith J.C. Greer J.J. Liu G.S. Feldman J.L. J. Neurophysiol. 1990; 64: 1149-1169Crossref PubMed Scopus (256) Google Scholar, 37Greer J.J. Smith J.C. Feldman J.L. J. Physiol. (Lond.). 1991; 437: 727-749Crossref Scopus (224) Google Scholar). The neuraxis was continuously perfused at 27 ± 1 °C (perfusion rate of 5 ml/min, chamber volume of 1.5 ml) with Kreb's solution that contained: 128 mm NaCl, 3.0 mm KCl, 1.5 mmCaCl2, 1.0 mm MgSO4, 24 mm NaHCO3, 0.5 mmNaH2PO4, and 30 mmd-glucose equilibrated with 95% O2, 5% CO2 at 27 °C (pH = 7.4). Details of the preparation have been described previously (38Smith J.C. Ellenberger H.H. Ballanyi K. Richter D.W. Feldman J.L. Science. 1991; 254: 726-729Crossref PubMed Scopus (1713) Google Scholar). Briefly, the brainstem/spinal cords isolated from the E18.5 fetuses were pinned down, ventral surface upward, on a paraffin-coated block. The block was mounted in the vise of a vibratome bath (Leica, VT1000S). The brainstem was serially sectioned in the transverse plane starting from the rostral medulla to within ∼100 mm of the rostral boundary of the pre-Bötzinger complex (38Smith J.C. Ellenberger H.H. Ballanyi K. Richter D.W. Feldman J.L. Science. 1991; 254: 726-729Crossref PubMed Scopus (1713) Google Scholar), as judged by the appearance of the inferior olive. A single transverse slice containing the pre-Bötzinger complex and more caudal reticular formation regions was then cut (300–400 μm thick), transferred to a recording chamber, and pinned down onto a Sylgard elastomer. The medullary slice was continuously perfused in physiological solution similar to that used for the brainstem/spinal cord preparation except for the potassium concentration, which was increased to 9 mm to stimulate the spontaneous rhythmic respiratory motor discharge in the medullary slice. Recordings of diaphragm electromyography (see Fig. 4), hypoglossal (XII) cranial nerve root (see Fig. 5), and population neuronal discharge within the pre-Bötzinger complex (see Fig. 5) were made with suction electrodes. Signals were amplified, rectified, low-passed filtered, and recorded on computer using an analog-digital converter (Digidata 1200, Axon Instruments) and data acquisition software (Axoscope, Axon Instruments). Mean values relative to control for the period and peak integrated amplitude of respiratory motoneuron discharge were calculated.Figure 5Recordings of integrated and rectified inspiratory neuronal discharge in medullary slice preparations isolated from E18.5 fetuses. As shown in A, the abnormal rhythmic patterns were also observed in recordings from the hypoglossal nerve rootlets of the medullary slice preparations fromSMIT1(−/−) fetuses. PBC, pre-Bötzinger complex. As shown in B, direct recordings of neuronal population discharge within the PBC demonstrated that the irregular rhythms were present in the putative respiratory rhythm generating center. As shown in C, as was the case in the brainstem/spinal cord preparation, the duration and amplitude of rectified and integrated motor discharge (other than augmented bursts) were decreased as compared with (+/+) preparation (recordings from XII motoneurons).View Large Image Figure ViewerDownload (PPT) Using a genomic clone containing the entire coding region of the murine SMIT1 gene (33McVeigh K.E. Mallee J.J. Lucente A. Barnoski B.L. Wu S. Berry G.T. Cytogenet. Cell Genet. 2000; 88: 153-158Crossref PubMed Google Scholar), we prepared a targeted deletion construct of the murine SMIT1gene (Fig. 1) and generated a homozygous deletion model. To obtain homozygous SMIT1 mutant (−/−) mice, heterozygous F1 females and males were mated, and genotypes of their F2 offspring were analyzed at postnatal day 21. Of 170 F2 animals collected, 63 of them were SMIT1(+/+), and 107 wereSMIT1(+/−). However, no SMIT1(−/−) mice were detected. We subsequently determined that the SMIT1(−/−) mice die shortly after birth. To determine the time of death of theSMIT1(−/−) offspring of (+/−) × (+/−) matings, cesarean sections were performed on E18.5 pregnant females. To confirm that the fetuses were alive at the time of Cesarean section, each fetus in the litter was stimulated by a gentle pinch with blunt end forceps before removal of the uterus. All responded to this physical stimulation by moving and extending extremities. By 20 min after the Cesarean birth, (+/+) and the (+/−) fetuses had begun to breath, move, squeal, and turn pink, whereas (−/−) pups became motionless and cyanotic following an interval characterized by irregular gasps of breath. All of the knock-out animals that were observed died within 20 min after birth. Body weight and external features of the newborn (−/−) fetuses were normal. In pathological examinations, there were no gross or microscopic malformations. Routine hematoxylin/eosin-stained sections of brain, spinal cord, dorsal root ganglia, heart, lungs, liver, kidneys, esophagus, stomach, intestines, adrenal, thyroid glands, and placenta appeared normal by light microscopy (data not shown). The deletion of the bulk of the SMIT1 coding region was confirmed by absent levels of SMIT1 transcript, demonstrated by Northern blot analyses of total RNA from fetal and placental samples (Fig.2). In a survey of total RNA from placenta and adult mouse tissues, the primary 11-kb SMIT1 transcript was most abundant in kidney, placenta, and brain with weak expression in thymus, lung, bladder, and testes (data not shown). In a survey of poly(A) RNA-enriched samples from adult brain and kidney and placental and embryo tissues, only an 11-kb transcript was detected (data not shown). The SMIT1 11-kb transcript, demonstrated by Northern blot analyses of total RNA from adult brain, was reduced in the heterozygous (+/−) mice as compared with wild-type (+/+) (Fig. 2). Using stable isotope dilution gas chromatography/mass spectrometry with hexadeuterated Ins ([2d6]Ins) in an isotope dilution analysis, we measured the levels of Ins in fetal brain, as well as whole embryos or fetuses obtained by cesarean section. At E10.5, E14.5, and E18.5, the total body Ins content inSMIT1(+/+) controls was 2.96 ± 0.45 (n= 8), 1.50 ± 0.13 (n = 3), and 1.16 ± 0.32 (n = 9) μmol/gram of wet weight, respectively (Fig.3A). In age-matchedSMIT1(−/−) mutants, the levels were reduced by 77% (n = 7), 64% (n = 6), and 84% (n = 3), respectively, whereas inSMIT1(+/−) heterozygotes, the levels were reduced by 52% (n = 5), 32% (n = 6), and 43% (n = 6), respectively. At E18.5, the Ins in isolated brain tissue was 7.80 ± 0.80 (n = 7), 5.98 ± 1.50 (n = 5), and 0.60 ± 0.40 (n = 3) μmol/grams of wet weight fromSMIT1(+/+), (+/−), and (−/−), respectively (Fig.3B). A similar trend was noted in amniotic fluid where the level of Ins was 480(n = 5), 325(n = 5), and 198 μm(n = 1) in theSMIT1(+/+), (+/−), (−/−) samples, respectively. In tissues obtained from control adult mice and analyzed by GC/MS, the level of Ins in brain, kidney, and liver was 4.31 ± (n = 3), 4.26 ± 1.34 (n = 3) and 0.10 ± 0.03 μmol/grams of wet weigh
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