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Ca2+ exchanger expression in the developing rat cortex
PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 0 5 9 - 3
Neuroscience Vol. 112, No. 1, pp. 65^73, 2002 C 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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Naþ /Ca2þ EXCHANGER EXPRESSION IN THE DEVELOPING RAT CORTEX G. T. GIBNEY,a J. H. ZHANG,a R. M. DOUGLAS,a G. G. HADDADa;b and Y. XIAa a
Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA b
Department of Cellular and Molecular Physiology, Yale University, New Haven, CT 06520, USA
Abstract4The Naþ /Ca2þ exchanger (NCX) participates in the regulation of neuronal Ca2þ homeostasis and is also believed to be involved in the neuronal responses to hypoxia. However, there are very limited data on how NCX mRNA and protein expression are regulated during brain development. In the present study, we sought to elucidate the developmental expression of NCX1 and NCX2 in the rat cortex from late fetal to adult stages using reverse transcription-polymerase chain reaction and western blot assays. The primers for NCX1 mRNA targeted the alternative splicing domain to allow di¡erentiation between NCX1 splice variants. Our results show that: (1) only two NCX1 mRNA splice variants (NCX1.5 and NCX1.4) are present in the cortex and their expression is age-dependent; (2) total NCX1 mRNA levels are low in fetal tissue, reach maximum density at postnatal day 8 and substantially decline with further maturation; (3) NCX2 mRNA density is signi¢cantly greater than total NCX1 mRNA for all ages and increases markedly during maturation from fetus/neonate to adult; and (4) NCX1 protein expression is lowest in late fetal cortex and reaches maximum levels after 2 weeks postnatally, even though expression levels are not signi¢cantly di¡erent between newborn and adult animals. Also, we found a similar NCX1 protein trend in the subcortical and cerebellar regions during development. From these data we suggest that NCX1 and NCX2 are di¡erentially expressed in the cortex with a predominance of NCX2 levels during postnatal development. We speculate that the developmental increase in NCX2 expression is responsible for the overall increase in Naþ /Ca2þ exchange capacity during maturation. C 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: NCX, Naþ /Ca2þ exchanger, development, cortex, brain, neurons.
Ca2þ ions play a signi¢cant role in neuronal communication. At the synapse, for example, calcium in£ux in the nerve terminal is responsible for neurotransmitter release into the synaptic cleft. Also, intracellular signaling mechanisms utilize Ca2þ ions as second messengers for such pathways as protein kinase regulation and NO synthase activation (Oancea and Meyer, 1998; Sugiya et al., 1998; Sola et al., 1999; Teubl et al., 1999). Therefore, regulation of Ca2þ ion homeostasis is a crucial factor in neuronal function and viability. The Naþ /Ca2þ exchanger (NCX) participates in this regulation via an exchange of three Naþ ions for one Ca2þ ion. Depending on cell state and electrochemical gradients, the NCX can function in either direction to achieve homeostasis (Blaustein and Lederer, 1999). It is expressed in a wide range of tissues and cell types, including heart, brain, kidney, and skeletal muscle (Lee
et al., 1994; Quednau et al., 1997). Past investigations have demonstrated the existence of three di¡erent NCX isoforms (NCX1, NCX2 and NCX3) with multiple splice variants of NCX1 (Aceto et al., 1992; Kofuji et al., 1994; Li et al., 1994; Nicoll et al., 1996). Furthermore, studies into the regional distribution of the NCX have revealed that the expression of the various isoforms and splice variants is tissue-speci¢c (Lee, 1994; Quednau et al., 1997; Yu and Colvin, 1997). In the mammalian adult brain, all three NCX isoforms are di¡erentially expressed with a predominance of NCX2 (Yu and Colvin, 1997). Immunolabeling experiments have shown that NCX is present in both neurons and astrocytes, with high densities localized on the presynaptic termini of nerve cells (Reuter and Porzig, 1995; Juha¤szova¤ et al., 1996; Ste¡ensen et al., 1997). At this synaptic region, NCX participates in Ca2þ e¥ux following signal transduction, as well as in ionic homeostasis throughout neurons (Ste¡ensen et al., 1997; Blaustein and Lederer, 1999). In addition to participating in normal cell function, NCX is also believed to be at least partially involved in hypoxia-induced intracellular Ca2þ overload, leading to neuronal injury (Stys et al., 1991; LoPachin and Lehning, 1997; Schroder et al., 1999). Since cortical neurons become increasingly vulnerable to hypoxic stress with age (Friedman and Haddad, 1993; Haddad and Jiang, 1993) and such stress-induced injury may cause
*Corresponding author. Tel. : +1-203-785-6101; fax: +1-203-7371252. E-mail address: [email protected] (Y. Xia). Abbreviations : DEPC, diechypyrocarbonate; E, embryonic day; GAPDH, glyceraldehyde-3-phosphate dehydrogenase ; NCX, Naþ /Ca2þ exchanger; P, postnatal day; RT-PCR, reverse transcription-polymerase chain reaction; TTBS, Tris-bu¡ered saline with Tween 20. 65
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serious neurological diseases (Mutch et al., 1992; Schwartzkroin et al., 1995), understanding how NCX expression is regulated in the cortex during development becomes important in elucidating the role of NCX during O2 deprivation. However, few studies have investigated the developmental expression of NCX in the brain, with only qualitative observations of mRNA and protein in the cerebellum (Li et al., 2000) and limited mRNA data in the cortex (Sakaue et al., 2000). Therefore, it remains unknown how NCX protein is developmentally expressed in the cortex and other brain regions, as well as whether di¡erent NCX1 splice variants are expressed in the cortex and vary with age. In the present study, we compared the developmental expression of NCX2 mRNA and NCX1 splice variant mRNA in the cortex. We also compared the developmental changes in NCX1 mRNA and protein since a speci¢c antibody for NCX1 protein was commercially available.
EXPERIMENTAL PROCEDURES
Animals Sprague^Dawley rats purchased from Charles River Laboratories (Wilmington, MA, USA) were used. Depending on the age group tested, pregnant rats were either killed for in-utero studies or allowed to give birth and tissues were harvested for studies. Prenatal and postnatal expression of mRNA and protein was investigated from embryonic day 16 (E16) through postnatal day 105 (P105). All animal procedures were performed in accordance with the guidelines of the Animal Care Committee of Yale University School of Medicine. Tissue preparation After anesthetization with methoxy£urane (Metofane; Pittman-Moore, Mundelein, IL, USA), each rat was decapitated, followed by removal of the brain from the cranium. The super¢cial lobes of the cerebrum were isolated, yielding the cortical tissue. The cerebellum was then removed and the remaining components, which were comprised of the diencephalon and brainstem, were saved as subcortical tissue. For mRNA studies, the cortex was frozen in liquid nitrogen and stored at 380‡C. In protein studies, the tissue samples were placed into ice-cold lysis bu¡er (200 mM mannitol, 80 mM HEPES (N-(2hydroxyethyl)piperazine-NP-(2-ethanesulphonic acid)), and 41 mM KOH, pH 7.4), containing a protease inhibitor cocktail (1 WM pepstatin A, 1 WM leupeptin, 230 WM phenylmethylsulfonyl £uoride, and 1 mM ethylenediamine tetrahydrochloride (Sigma, St. Louis, MO, USA)). For the embryonic and newborn (P1) groups, six to eight samples were pooled together to provide a su⁄cient amount of tissue for mRNA and protein analysis. Individual brain regions were tested in all other sample groups. RNA extraction Total RNA was extracted from the cortical samples using Trizol reagent (Gibco-BRL, Life Technologies, Rockville, MD, USA) according to the manufacturer’s protocol, which is based on the method of Chomcynski and Sacchi (1987). In brief, samples were homogenized in Trizol reagent and incubated at room temperature for 5 min to allow complete dissociation of nucleoprotein complexes. Chloroform (200 Wl/ml Trizol) was added and samples were centrifuged, followed by removal of the aqueous phase. RNA was precipitated using isopropanol (0.5 ml isopropanol/ml Trizol), centrifuged, washed with ethanol and resuspended in diechypyrocarbonate (DEPC)-treated water.
Membrane protein preparation The isolated tissue samples were removed from the lysis bu¡er solution and transferred to 4Uvolume/weight of lysis bu¡er. The tissue was homogenized by 10^20 strokes with a Te£onglass homogenizer (Thomas Scienti¢c, Swedesboro, NJ, USA) at 2000 r.p.m. After that, the homogenate was centrifuged for 10 min at 1000Ug. The supernatant was then removed and centrifuged again for 1 h at 100 000Ug. Upon completion, the pellet was resuspended in 200^1000 Wl of lysis bu¡er and stored at 380‡C for future use. Reverse transcription-polymerase chain reaction (RT-PCR) measurement Complementary DNA (cDNA) was synthesized by RT from 2 Wg of total RNA for 1 h at 37‡C and for 5 min at 95‡C using 100 ng of poly dT15 primer (Boehringer Mannheim, Mannheim, Germany) and 20 units of reverse transcriptase (M-MuLV; Boehringer Mannheim) in bu¡ered DEPC water (20 Wl total volume). PCR was carried out for NCX1, NCX2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) utilizing speci¢c primers we designed. Their respective sequences were 5P-AAC ACT GCC ACC ATA ACC-3P and 5P-CTC CCC ACA TTC ATC ATC-3P [NCX1, bases 1689^2381 (Furman et al., 1993)], 5P-GAC GAC GAA GAG TAT GAG AAG-3P and 5P-AGT GAG CAG ACC AAT GAC C-3P [NCX2, bases 1789^2289 (Li et al., 1994)], and 5P-ACC ACC ATG GAG AAG GCT GG-3P and 5P-AAC ACG GAA GGC CAT GCC AG-3P [GAPDH, bases 310^729 (Tso et al., 1985)]. All three transcripts were ampli¢ed simultaneously in separate reaction vessels with their respective forward and reverse primers in the presence of 2.5 units of RED Tag polymerase (Sigma). In initial experiments, multiple volumes (0, 2.5, 5 and 10 Wl) for the RT products were sampled to determine an optimal volume for each assay. A volume of 5 Wl was then chosen for all developmental samples. Ampli¢cation was also carried out for various cycle numbers (25, 30 and 35 cycles), each consisting of 1 min at 60‡C, and 1.5 min at 72‡C, and 1 min at 95‡C, using a BioRad iCycler (Bio-Rad, Hercules, CA, USA). Thirty cycles was deemed optimal and was used in subsequent experiments. All PCR products were visualized via gel electrophoresis (1.5% agarose gel) and ethidium bromide staining. Densitometric analysis of the band densities was done with ImageQuanNT software (Molecular Dynamics, Sunnyvale, CA, USA). Sequencing Since two bands were present after gel electrophoresis of the NCX1 PCR product, their respective sequences were determined to ensure that both were ampli¢cations of NCX1 cDNA. Once the bands were su⁄ciently separated by electrophoresis, both products were isolated following the QIAquick Gel Extraction Kit Protocol using a vacuum manifold (Qiagen, Valencia, CA, USA). In brief, each band was excised from the gel and dissolved in QG bu¡er (300 Wl/100 mg of gel). Isopropanol (100 Wl/100 mg of gel) was then added, and the mixture was placed in the QIAquick column with vacuum. The column was rinsed ¢rst with 0.5 ml of QG bu¡er and then 0.75 ml of PE bu¡er. Subsequently, 50 Wl of H2 O was added to the column and centrifuged at 10 000Ug. The water sample, containing the puri¢ed PCR product, was sent to the W.M. Keck Foundation Biotechnology Resource Lab (Yale University School of Medicine) for sequencing. Using dideoxynucleotide chain termination reaction and automated detection (ABI Prism Model 3700; Applied Biosystems, Applera Corporation, Foster City, CA, USA), the precise sequences for both cDNA segments were determined. Western blotting Protein concentrations of each sample were determined using a DC Protein Assay Kit (Bio-Rad). 25 Wg protein samples in Tris-bu¡ered loading solution (2% sodium dodecyl sulfate, 100 mM dithiothreitol, 10% glycerol, 0.02% Bromophenol Blue, and
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20 mM Tris, pH 6.8) were electrophoresed in 10% polyacrylamide gels and transferred to polyvinylidene £uoride membranes (Immobilon-P, Millipore, Bedford, MA, USA) at 350 mA for 3 h at 4‡C. The transfer of protein to the membranes was assessed by Ponceau S staining. Membranes were blocked in Tris-bu¡ered saline with Tween 20 (TTBS) bu¡er (0.1% Tween 20 (Bio-Rad); 20 mM Tris, 500 mM NaCl) with 5% non-fat dry milk (Carnation, Nestle Food Co., Glendale, CA, USA) for 1 h, followed by three rinse cycles in TTBS. Membranes were then incubated with the rabbit anti-NCX1 antibody (1:1000; RBI, Natick, MA, USA and SWANT, Switzerland) in TTBS and 2.5% milk for 3 h. After three rinses in TTBS, membranes were incubated with secondary antibody, anti-rabbit immunoglobulin G (IgG) linked to horseradish peroxidase (1:1000; Zymed, South San Francisco, CA, USA) in TTBS and 2.5% milk for 1 h. Signal detection was carried out using the ECL chemiluminescence system (Amersham, Little Chalfont, UK). All membranes were stripped, and re-probed with a mouse
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monoclonal antibody to L-actin (Chemicon, Temecula, CA, USA). Densitometric analysis of the immunoblot ¢lms was conducted on a Personal Densitometer SI scanner and ImageQuaNT analysis software (Molecular Dynamics). Data analysis mRNA measurements were ¢rst normalized to GAPDH and then expressed as a percentage of the respective P23 group, while NCX1 protein measurements were normalized to total protein and expressed as a percentage of the respective P23 density located on the same membrane. This P23 group was chosen rather than the P30 group due to the limited expression observed in NCX1 mRNA at P30. All data are represented as mean X S.E.M. and were subjected to statistical analysis with the Student’s t-test. Data analysis was performed using GraphPad Prism 3.0 software (GraphPad Software, Inc., San Diego, CA, USA).
Fig. 1. Speci¢city of RT-PCR ampli¢cation. (A) Gel electrophoresis of the NCX1 and NCX2 RT-PCR products with a 100 bp DNA ladder is shown. NCX1 and NCX2 products were ampli¢ed from the same RT samples and electrophoresed through a 1.5% agarose gel. The predicted sizes for the NCX1 ampli¢cation products were 693 bp and 627 bp, while ampli¢cation of the NCX2 isoform resulted in a single 501 bp product. (B) cDNA sequence comparison of the two NCX1 bands is shown. After sequencing, the NCX1 bands were identi¢ed as identical sequences with the exception of a 66 bp omission in the lower band, indicated by the non-shaded area. Further analysis identi¢ed them as splice variants of the NCX1 gene, yielding the notation NCX1-V1 and NCX1-V2.
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G. T. Gibney et al. RESULTS
Speci¢city of NCX primers and isoform identi¢cation Two sets of primers were used for cDNA ampli¢cation of NCX1 and NCX2 in the rat cortex. The primers chosen for NCX1 were speci¢cally designed to amplify the variable region of the NCX1 mRNA transcript where alternative splicing occurs. This allowed quanti¢cation of both total NCX1 mRNA and individual NCX1 splice variants. Gel electrophoresis revealed the presence of two di¡erent PCR products with NCX1 primers, but only a single band from NCX2 ampli¢cation (Fig. 1A). Subsequent sequencing of the NCX1 products showed that the two transcripts shared 97^98% sequence similarity with the previously reported rat brain NCX1 sequence (Furman et al., 1993). As represented in Fig. 1B, the smaller NCX1 band, designated NCX1-V2, differs from the larger NCX1 band, designated NCX1-V1, by a 66 bp deletion from bases 2049^2116. Also of signi¢cance is that the predicted sizes of the NCX1 and NCX2 bands correspond well to the observed sizes determined from gel electrophoresis with the 100 bp DNA marker (Fig. 1A). RT dilution and cycle optimization In order to determine the optimal conditions for the polymerase chain reaction assays, various RT product volumes and cycle numbers were utilized for samples at P1 and P23. These ages were chosen since our preliminary data revealed that signi¢cant developmental di¡erences in expression existed for NCX and it would be necessary to incorporate this factor into our experimental design. As shown in Fig. 2A, three RT volumes (2.5, 5 and 10 Wl), as well as a negative control, were assayed. The 10 Wl lanes appear to approach signal saturation when compared to the 5 Wl lanes, especially in NCX2. Since the band density at 2.5 Wl was either faint or not present, a volume of 5 Wl for the RT product was optimal. Also, it should be noted that no bands were present in negative controls (0 Wl lanes), suggesting that our experiments were not contaminated by any source of exogenous RNA. In terms of cycle optimization (Fig. 2B), 30 cycles yielded the best ampli¢cation product for NCX1, NCX2 and GAPDH. After 35 cycles, NCX2 band density was over-saturated and demonstrated some non-speci¢c ampli¢cation, while GAPDH intensity remained constant or slightly decreased in comparison to 30 cycles. With only 25 cycles, no bands or only faint bands were observed in each group. Based on these experiments, it was determined that an RT volume of 5 Wl and a cycle number of 30 were optimal for measuring the developmental expression of NCX. Expression of NCX1 mRNA in the developing cortex The expression of NCX1 mRNA (NCX1-V1 and NCX1-V2) in the developing cortex was determined at six di¡erent ages, i.e. E22, P4, P8, P13, P23, and P30. RT-PCR was conducted three times for each RNA sam-
Fig. 2. Optimization experiments for RT-PCR assays. (A) Gel electrophoresis of NCX1 and NCX2 RT-PCR products for di¡erent RT volumes (RT: 0, 2.5, 5 and 10 Wl) is shown. A volume of 0 Wl RT product was used as a negative control. In both NCX1 and NCX2 assays, 2.5 Wl of RT product yielded weak band signals after PCR ampli¢cation, whereas band densities from 10 Wl volumes were largely over-saturated. (B) Gel electrophoresis of NCX1, NCX2 and GAPDH RT-PCR products from di¡erent cycles (25 cycles: C25; 30 cycles: C30; 35 cycles : C35) is shown. The band densities from 25 cycles of PCR ampli¢cation were weak or below detection levels, while band densities from 35 cycles were over-saturated. Note that P1 and P23 age groups were assessed for optimal conditions since NCX expression varies substantially during development.
ple, and the individual values from each assay were averaged together to ensure accurate assessment. Each age group from di¡erent six cortex samples represents the average NCX1 mRNA density. Total NCX1 mRNA expression was relatively low in the youngest age group and rose dramatically to a peak level at P8 (Fig. 3A, B). Beyond this age, NCX1 mRNA levels steadily decreased, reaching a minimal density level at P30. The validity of these data was supported by the relatively constant expression levels of GAPDH mRNA throughout development. In addition, the NCX1 splice variants were differentially expressed (Fig. 3C). Both NCX1-V1 and NCX1-V2 were initially present in approximately equal amounts. With increasing age, the proportion of NCX1V1 mRNA steadily increased, while that of NCX1-V2 steadily decreased. At P30 and thereafter, NCX1-V2 levels reached undetectable levels, leaving only NCX1-V1 present at this age.
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was applied as was conducted for NCX1 measurements. The results showed a pattern of expression that was dissimilar to that of NCX1 mRNA expression (Fig. 4). Initially, the relative expression of NCX2 mRNA was low during early development (embryo to neonate). This level substantially increased by P8, reaching a plateau thereafter. Furthermore, di¡erences in expression levels between NCX1 and NCX2 at various ages could be analyzed, since both NCX isoforms were individually ampli¢ed from the same RT products. The expression level of NCX2 mRNA was found to be 2 to 4.5-fold greater than the level of total NCX1 mRNA present in each age group, even though the band size of NCX2 (501 bp) was smaller than either NCX1 band sizes (693 and 627 bp). For example, the average density of total NCX1 normalized to GAPDH at P8 was 0.162 units, whereas that of NCX2 was 0.466 units, a three-fold di¡erence in expression levels. NCX1 protein expression in the cortex and other brain regions during development To determine the developmental expression of NCX1 protein, western blot assays were performed using a speci¢c anti-NCX1 antibody (Fig. 5), which crossreacts with
Fig. 3. NCX1 mRNA expression during development of the cortex. (A) Gel electrophoresis of the NCX1 RT-PCR products at ages P4, P8, P13 and P23 is shown. (B) The developmental pro¢le of total NCX1 mRNA expression for ages E22 through P30 is shown. Densitometric measurements of the NCX1 RT-PCR products were normalized to GAPDH and then calculated as a percentage of their respective P23 groups. Each group is represented as the mean X S.E.M. of six individual samples. P values: P = 0.01, P4 vs P23, P8 vs P23, P13 vs P30; P 6 0.001, P4 vs P30, P8 vs P30, P23 vs P30. The /embryonic group was not compared to other groups since the data were obtained from 2 pooled samples (6^8 tissues per pool). Note that NCX1 exhibits low expression during early development, reaches maximal levels at P8 and declines thereafter. (C) The di¡erential expression of NCX1 splice variants for each age group is shown. Data are represented as the mean X S.E.M. of the percentage of total NCX1 product density for each age group. The asterisk (D) represents a statistical di¡erence (P 6 0.05) from NCX1-V1 density based on Student’s t-test analysis. Note that NCX1-V1 is the predominant variant expressed with further development of the cortex. Also, NCX1-V2 density in P30 samples was below detectable levels.
Expression of NCX2 mRNA in the developing cortex To study the developmental expression of NCX2 mRNA, PCR was carried out utilizing speci¢c NCX2 primers to amplify the reverse transcript of each sample. The same approach for accurate RT-PCR assessment
Fig. 4. NCX2 mRNA expression during development of the cortex. (A) Gel electrophoresis of the NCX2 RT-PCR product at ages P4, P8, P13 and P23 is shown. (B) The developmental pro¢le of NCX2 mRNA expression for ages E22 through P30 is shown. Densitometric measurements of the NCX2 RT-PCR products were normalized to GAPDH and then calculated as a percentage of their respective P23 groups. Each group is represented as the mean X S.E.M. of six individual samples. P values: P 6 0.05, P4 vs P30; P 6 0.0001, P4 vs P8, P4 vs P13, P4 vs P23. The embryonic group was not subject to statistical comparison because the data were obtained from 2 pooled samples (6^8 tissues per pool). Note that NCX2 exhibits low expression in early ages, and increases to adult levels by P8.
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both NCX1 variants. Since NCX1 mRNA decreased substantially after P8^13, we extended our NCX1 protein measurements to P105 to determine if there was a subsequent decline in expression with further maturation. Also, P8 group was omitted since only slight changes existed in mRNA densities between successive ages. In the cortex, NCX1 protein levels were low in embryonic tissue (E16^18: 40% of P23) and rose signi¢cantly to a peak level by P13 (Fig. 5A, B). After P13, NCX1 protein density declined, yielding a trend that parallels the mRNA results. With further maturation from P30 to P105, the level of NCX1 protein continued to decline. To determine whether the developmental pattern of NCX1 protein is cortex-speci¢c, we further compared
NCX1 protein expression in the cortex to that in the brainstem/diencephalon and cerebellum. As was observed in the developing cortex, NCX1 protein expression in the subcortex was lowest in the embryonic age group (E16^18: 35% of P23) and increased steadily through P13 (110% of P23) (Fig. 5C, D). With age, NCX1 expression decreased to adult levels. After 105 days of postnatal growth, NCX1 protein expression decreased to levels comparable to those of newborn NCX1 densities (P105: 65% of P23). In contrast, NCX1 protein expression in the cerebellum did not statistically change during development, although there was a tendency to decrease with age (Fig. 5E, F). From newborn to P23, NCX1 density remained constant with only
Fig. 5. Developmental pro¢le of NCX1 protein expression. Western blots of NCX1 at ages E16, P1, P13, P23, P30 and P105 are shown for the cortical (A), subcortical (C) and cerebellar (E) regions. 25 Wg of total protein was loaded onto 10% polyacrylamide gels, separated by sodium dodecyl sulfate^polyacrylamide gel electrophoresis for 1 h at 100 mV and transferred to polyvinylidene £uoride membranes. Membranes were probed with a highly speci¢c NCX1 antibody. Densitometric measurements of NCX1 band densities at ages E16 through P105 are shown for cortical (B), subcortical (D) and cerebellar (F) regions. Measurements were normalized to total membrane protein and expressed as a percentage of the P23 group. Each group is represented as mean values X S.E.M. from three samples. P values in B (cortex): P 6 0.05, E vs P13, E vs P23, P13 vs P105, P23 vs P105; P = 0.001, P13 vs P30, P23 vs P30; P 6 0.0001, P13 vs P23. P values in D (subcortex) : P 6 0.05, E vs P1, E vs P13, E vs P30, P1 vs P13, P13 vs P23, P30 vs P105; P = 0.005, P1 vs P105; P 6 0.001, E vs P23, P1 vs P23, P13 vs P105; P 6 0.0001, P23 vs P105.
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slight deviations (P1: 102% of P23; P13: 90% of P23). Beyond this stage of development, NCX1 expression tended to decline (P30: 95% of P23; P105: 70% of P23). Note that cerebellar NCX1 protein density was not measured in the E16^18 age group because the limited size of the cerebellum was not su⁄cient for protein assessment.
DISCUSSION
In the present study we have made several important observations pertaining to the NCX in the developing rat cortex: (1) NCX1 splice variant mRNA is di¡erentially expressed depending on age; (2) NCX1 and NCX2 mRNA expression increases from fetus to P8, but diverges thereafter with a substantial decline in NCX1 density and a constant level of NCX2 density; and (3) in spite of its low density in the fetus, NCX1 protein expression remains at a relatively constant level from newborn to adult. Di¡erential splicing of NCX1 during cortical development Within the alternative splicing region of the NCX1 mRNA isoform, there are six identi¢able cassette exons, designated exons A, B, C, D, E and F (Kofuji et al., 1994; Quednau et al., 1997). Although there have been as many as nine di¡erent NCX1 splice variants identi¢ed in adult rat tissues, only three distinct splicing combinations (ACD, AD and ADF) have been observed in the brain (Kofuji et al., 1994; Quednau et al., 1997). In this work, we utilized speci¢c primers to detect all variants and found that only two NCX1 splice variants, NCX1-V1 and NCX1-V2, are present in the developing cortex. NCX1-V1 contained exons ADF and NCX1-V2 contained exons AD, which correspond to the splice variant notation NCX1.5 and NCX1.4 (Quednau et al., 1997), respectively. Interestingly, the exclusion of the short cassette exon F was found to di¡er between ages. With maturation of the cortex, the proportion of NCX1-V1 mRNA in relation to total NCX1 mRNA steadily increased, while the proportion of NCX1-V2 vastly declined, demonstrating that the expression of NCX1 splice variants is age-dependent. The di¡erential regulation of NCX1 splice variant expression may be of signi¢cance in neuronal function. Past investigations have demonstrated that NCX1.4 responds di¡erently to applied physiological conditions as compared to splice variants NCX1.1 and NCX1.3 (Matsuoka et al., 1995; Dyck et al., 1999). NCX1.5 and NCX1.4, the two splice variants observed in this study, have not been subjected to functional comparison. However, the inclusion/exclusion of exon F may yield a functional di¡erence between these variants. So far, multiple sites for possible phosphorylation via protein kinases have been identi¢ed on the cytoplasmic loop (Pearson and Kemp, 1991; Matsuda et al., 1997). This F exon lies adjacent to consensus regulatory domains and contains three serine residues (Quednau et al., 1997) that may be potential sites for phosphorylation.
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Moreover, such regulation has been observed in vascular smooth muscle cells where the phosphorylation of multiple serine residues led to stimulation in NCX activity (Matsuda et al., 1997). If the inclusion of cassette exon F by NCX1.5 o¡ers a regulatory advantage over NCX1.4, neuronal development may selectively favor the expression of the former splice variant as maintenance of calcium homeostasis becomes increasingly important with further maturation (Jiang and Guro¡, 1997; Finkbeiner, 2000). Developmental changes in NCX mRNA and protein Little is known about the developmental expression of NCX subtypes in the cortex and other regions of the brain. Our semi-quantitative PCR data suggest that NCX1 and NCX2 mRNA are di¡erentially expressed in the developing cortex. Since constant GAPDH mRNA band densities were observed throughout the development assays, we are con¢dent that the disparity between NCX mRNA levels represents a speci¢c change in NCX expression during development. In addition to mRNA analysis, NCX1 membrane protein expression was assessed in the developing cortex, as well as diencephalon/brainstem and cerebellar regions. We chose not to normalize the NCX1 protein densities to L-actin levels because in this study, and in other recent work by our group, we have found that L-actin membrane protein density is not constant in various brain regions throughout development (Douglas et al., 2001). This developmental alteration in L-actin expression may be related to either an increase in neuronal populations that occurs in postnatal rat development or to an increase in overall protein density per cell (Douglas et al., 2001). With either event occurring, proteins that are normally constitutively expressed may not serve as stable reference points for extended developmental periods, such as those analyzed in this study. Therefore, NCX1 protein densities were presented to show relative changes between ages, while L-actin protein levels were utilized for the assessment of general protein loading and membrane transfer e⁄cacy of the western blots, as we have done in previous investigations (Douglas et al., 2001). So far it is not clear as to which NCX isoforms signi¢cantly contribute to the increase in Naþ /Ca2þ exchange capacity with maturation of the mammalian brain (Juha¤szova¤ and Rus›e'a¤k, 1991; Sakaue et al., 2000). Our experiments demonstrated that NCX1 and NCX2 mRNA pro¢les diverge after an early increase in expression, with a decline in NCX1 mRNA and constant NCX2 levels beyond P8. Also, NCX2 mRNA expression is signi¢cantly greater than that of NCX1 mRNA in all age groups. This clearly indicates that NCX2 plays a dominant role in Naþ /Ca2þ exchange at all ages. The elevated expression of NCX2 may account for the observed developmental increase in ionic exchange. This is further supported by our NCX1 protein expression data in demonstrating no signi¢cant difference between newborn and adult levels. One of the mechanisms of hypoxic injury via intracel-
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lular Ca2þ overload is believed to involve ‘reverse mode operation’ of NCX (Stys et al., 1991; LoPachin et al., 1997). Indeed, the non-speci¢c NCX inhibitor, KBR7943, has been shown to protect CA1 neurons from hypoxia-induced injury (Schroder et al., 1999). However, it is unclear whether developmental changes in the NCX expression correspond to di¡erences in hypoxic susceptibility between newborn and adult tissues. Our results show that NCX1 mRNA levels are higher in the newborn than in adult and that NCX1 protein expression is approximately equal in the immature and mature brain, including the cortical, subcortical and cerebellar regions. These data suggest that NCX1 expression may not be directly related to age-dependent susceptibility. On the contrary, NCX2 mRNA increased with age and maintained constant levels with further development. Whether its developmental expression is related to the increase in hypoxic susceptibility with age is not clear at present.
In conclusion, our ¢ndings have elucidated the developmental expression patterns of NCX1 and NCX2 in the rat cortex. Our results suggest that NCX2 may play a predominant role in Naþ /Ca2þ exchange in the developing and mature cortex, while NCX1 protein expression does not substantially change between newborn and adult levels. Since NCX has been linked to hypoxia-induced Ca2þ overload (Stys et al., 1991; LoPachin et al., 1997), further study is important to determine whether this developmental increase in NCX2 expression is related to the observed di¡erences in hypoxic susceptibility between immature and mature neurons.
Acknowledgements/This work was supported by March of Dimes (FY00-722) and NIH (R01 HD-34852) to Y.X. and NIH grants (PO1 HD-32573 and RO1 NS-35918) to G.G.H.
REFERENCES
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Sola, C., Barron, S., Tusell, J.M., Serratosa, J., 1999. The Ca2þ /calmodulin signaling system in the neuronal response to excitability. Involvement of neuronal and glial cells. Prog. Neurobiol. 58, 207^232. Ste¡ensen, I., Waxman, S.G., Mills, L., Stys, P.K., 1997. Immunolocalization of the Naþ -Ca2þ exchanger in mammalian myelinated axons. Brain Res. 776, 1^9. Stys, P.K., Waxman, S.G., Ransom, B.R., 1991. Naþ -Ca2þ exchanger mediates Ca2þ in£ux during anoxia in mammalian central nervous system white matter. Ann. Neurol. 30, 375^380. Sugiya, H., Michikawa, H., Mitsui, Y., Fujita-Yoshigaki, J., Hara-Yokoyama, M., Furuyama, S., 1998. Ca2þ -nitric oxide-cGMP signaling in rabbit parotid acinar cells. Eur. J. Morphol. 36, 194^197. Teubl, M., Groschner, K., Kohlwein, S.D., Mayer, B., Schmidt, K., 1999. Naþ /Ca2þ exchange facilitates Ca2þ -dependent activation of endothelial nitric-oxide synthase. J. Biol. Chem. 274, 29529^29535. Tso, J.Y., Sun, X.H., Kao, T.H., Reece, K.S., Wu, R., 1985. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs : genomic complexity and molecular evolution of the gene. Nucleic Acids Res. 13, 2485^2502. Yu, L., Colvin, R.A., 1997. Regional di¡erences in expression of transcripts for Naþ /Ca2þ exchanger isoforms in rat brain. Brain Res. Mol. Brain Res. 50, 285^292. (Accepted 31 January 2002)
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Neuroscience Vol. 112, No. 1, pp. 65^73, 2002 C 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
Naþ /Ca2þ EXCHANGER EXPRESSION IN THE DEVELOPING RAT CORTEX G. T. GIBNEY,a J. H. ZHANG,a R. M. DOUGLAS,a G. G. HADDADa;b and Y. XIAa a
Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA b
Department of Cellular and Molecular Physiology, Yale University, New Haven, CT 06520, USA
Abstract4The Naþ /Ca2þ exchanger (NCX) participates in the regulation of neuronal Ca2þ homeostasis and is also believed to be involved in the neuronal responses to hypoxia. However, there are very limited data on how NCX mRNA and protein expression are regulated during brain development. In the present study, we sought to elucidate the developmental expression of NCX1 and NCX2 in the rat cortex from late fetal to adult stages using reverse transcription-polymerase chain reaction and western blot assays. The primers for NCX1 mRNA targeted the alternative splicing domain to allow di¡erentiation between NCX1 splice variants. Our results show that: (1) only two NCX1 mRNA splice variants (NCX1.5 and NCX1.4) are present in the cortex and their expression is age-dependent; (2) total NCX1 mRNA levels are low in fetal tissue, reach maximum density at postnatal day 8 and substantially decline with further maturation; (3) NCX2 mRNA density is signi¢cantly greater than total NCX1 mRNA for all ages and increases markedly during maturation from fetus/neonate to adult; and (4) NCX1 protein expression is lowest in late fetal cortex and reaches maximum levels after 2 weeks postnatally, even though expression levels are not signi¢cantly di¡erent between newborn and adult animals. Also, we found a similar NCX1 protein trend in the subcortical and cerebellar regions during development. From these data we suggest that NCX1 and NCX2 are di¡erentially expressed in the cortex with a predominance of NCX2 levels during postnatal development. We speculate that the developmental increase in NCX2 expression is responsible for the overall increase in Naþ /Ca2þ exchange capacity during maturation. C 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: NCX, Naþ /Ca2þ exchanger, development, cortex, brain, neurons.
Ca2þ ions play a signi¢cant role in neuronal communication. At the synapse, for example, calcium in£ux in the nerve terminal is responsible for neurotransmitter release into the synaptic cleft. Also, intracellular signaling mechanisms utilize Ca2þ ions as second messengers for such pathways as protein kinase regulation and NO synthase activation (Oancea and Meyer, 1998; Sugiya et al., 1998; Sola et al., 1999; Teubl et al., 1999). Therefore, regulation of Ca2þ ion homeostasis is a crucial factor in neuronal function and viability. The Naþ /Ca2þ exchanger (NCX) participates in this regulation via an exchange of three Naþ ions for one Ca2þ ion. Depending on cell state and electrochemical gradients, the NCX can function in either direction to achieve homeostasis (Blaustein and Lederer, 1999). It is expressed in a wide range of tissues and cell types, including heart, brain, kidney, and skeletal muscle (Lee
et al., 1994; Quednau et al., 1997). Past investigations have demonstrated the existence of three di¡erent NCX isoforms (NCX1, NCX2 and NCX3) with multiple splice variants of NCX1 (Aceto et al., 1992; Kofuji et al., 1994; Li et al., 1994; Nicoll et al., 1996). Furthermore, studies into the regional distribution of the NCX have revealed that the expression of the various isoforms and splice variants is tissue-speci¢c (Lee, 1994; Quednau et al., 1997; Yu and Colvin, 1997). In the mammalian adult brain, all three NCX isoforms are di¡erentially expressed with a predominance of NCX2 (Yu and Colvin, 1997). Immunolabeling experiments have shown that NCX is present in both neurons and astrocytes, with high densities localized on the presynaptic termini of nerve cells (Reuter and Porzig, 1995; Juha¤szova¤ et al., 1996; Ste¡ensen et al., 1997). At this synaptic region, NCX participates in Ca2þ e¥ux following signal transduction, as well as in ionic homeostasis throughout neurons (Ste¡ensen et al., 1997; Blaustein and Lederer, 1999). In addition to participating in normal cell function, NCX is also believed to be at least partially involved in hypoxia-induced intracellular Ca2þ overload, leading to neuronal injury (Stys et al., 1991; LoPachin and Lehning, 1997; Schroder et al., 1999). Since cortical neurons become increasingly vulnerable to hypoxic stress with age (Friedman and Haddad, 1993; Haddad and Jiang, 1993) and such stress-induced injury may cause
*Corresponding author. Tel. : +1-203-785-6101; fax: +1-203-7371252. E-mail address: [email protected] (Y. Xia). Abbreviations : DEPC, diechypyrocarbonate; E, embryonic day; GAPDH, glyceraldehyde-3-phosphate dehydrogenase ; NCX, Naþ /Ca2þ exchanger; P, postnatal day; RT-PCR, reverse transcription-polymerase chain reaction; TTBS, Tris-bu¡ered saline with Tween 20. 65
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serious neurological diseases (Mutch et al., 1992; Schwartzkroin et al., 1995), understanding how NCX expression is regulated in the cortex during development becomes important in elucidating the role of NCX during O2 deprivation. However, few studies have investigated the developmental expression of NCX in the brain, with only qualitative observations of mRNA and protein in the cerebellum (Li et al., 2000) and limited mRNA data in the cortex (Sakaue et al., 2000). Therefore, it remains unknown how NCX protein is developmentally expressed in the cortex and other brain regions, as well as whether di¡erent NCX1 splice variants are expressed in the cortex and vary with age. In the present study, we compared the developmental expression of NCX2 mRNA and NCX1 splice variant mRNA in the cortex. We also compared the developmental changes in NCX1 mRNA and protein since a speci¢c antibody for NCX1 protein was commercially available.
EXPERIMENTAL PROCEDURES
Animals Sprague^Dawley rats purchased from Charles River Laboratories (Wilmington, MA, USA) were used. Depending on the age group tested, pregnant rats were either killed for in-utero studies or allowed to give birth and tissues were harvested for studies. Prenatal and postnatal expression of mRNA and protein was investigated from embryonic day 16 (E16) through postnatal day 105 (P105). All animal procedures were performed in accordance with the guidelines of the Animal Care Committee of Yale University School of Medicine. Tissue preparation After anesthetization with methoxy£urane (Metofane; Pittman-Moore, Mundelein, IL, USA), each rat was decapitated, followed by removal of the brain from the cranium. The super¢cial lobes of the cerebrum were isolated, yielding the cortical tissue. The cerebellum was then removed and the remaining components, which were comprised of the diencephalon and brainstem, were saved as subcortical tissue. For mRNA studies, the cortex was frozen in liquid nitrogen and stored at 380‡C. In protein studies, the tissue samples were placed into ice-cold lysis bu¡er (200 mM mannitol, 80 mM HEPES (N-(2hydroxyethyl)piperazine-NP-(2-ethanesulphonic acid)), and 41 mM KOH, pH 7.4), containing a protease inhibitor cocktail (1 WM pepstatin A, 1 WM leupeptin, 230 WM phenylmethylsulfonyl £uoride, and 1 mM ethylenediamine tetrahydrochloride (Sigma, St. Louis, MO, USA)). For the embryonic and newborn (P1) groups, six to eight samples were pooled together to provide a su⁄cient amount of tissue for mRNA and protein analysis. Individual brain regions were tested in all other sample groups. RNA extraction Total RNA was extracted from the cortical samples using Trizol reagent (Gibco-BRL, Life Technologies, Rockville, MD, USA) according to the manufacturer’s protocol, which is based on the method of Chomcynski and Sacchi (1987). In brief, samples were homogenized in Trizol reagent and incubated at room temperature for 5 min to allow complete dissociation of nucleoprotein complexes. Chloroform (200 Wl/ml Trizol) was added and samples were centrifuged, followed by removal of the aqueous phase. RNA was precipitated using isopropanol (0.5 ml isopropanol/ml Trizol), centrifuged, washed with ethanol and resuspended in diechypyrocarbonate (DEPC)-treated water.
Membrane protein preparation The isolated tissue samples were removed from the lysis bu¡er solution and transferred to 4Uvolume/weight of lysis bu¡er. The tissue was homogenized by 10^20 strokes with a Te£onglass homogenizer (Thomas Scienti¢c, Swedesboro, NJ, USA) at 2000 r.p.m. After that, the homogenate was centrifuged for 10 min at 1000Ug. The supernatant was then removed and centrifuged again for 1 h at 100 000Ug. Upon completion, the pellet was resuspended in 200^1000 Wl of lysis bu¡er and stored at 380‡C for future use. Reverse transcription-polymerase chain reaction (RT-PCR) measurement Complementary DNA (cDNA) was synthesized by RT from 2 Wg of total RNA for 1 h at 37‡C and for 5 min at 95‡C using 100 ng of poly dT15 primer (Boehringer Mannheim, Mannheim, Germany) and 20 units of reverse transcriptase (M-MuLV; Boehringer Mannheim) in bu¡ered DEPC water (20 Wl total volume). PCR was carried out for NCX1, NCX2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) utilizing speci¢c primers we designed. Their respective sequences were 5P-AAC ACT GCC ACC ATA ACC-3P and 5P-CTC CCC ACA TTC ATC ATC-3P [NCX1, bases 1689^2381 (Furman et al., 1993)], 5P-GAC GAC GAA GAG TAT GAG AAG-3P and 5P-AGT GAG CAG ACC AAT GAC C-3P [NCX2, bases 1789^2289 (Li et al., 1994)], and 5P-ACC ACC ATG GAG AAG GCT GG-3P and 5P-AAC ACG GAA GGC CAT GCC AG-3P [GAPDH, bases 310^729 (Tso et al., 1985)]. All three transcripts were ampli¢ed simultaneously in separate reaction vessels with their respective forward and reverse primers in the presence of 2.5 units of RED Tag polymerase (Sigma). In initial experiments, multiple volumes (0, 2.5, 5 and 10 Wl) for the RT products were sampled to determine an optimal volume for each assay. A volume of 5 Wl was then chosen for all developmental samples. Ampli¢cation was also carried out for various cycle numbers (25, 30 and 35 cycles), each consisting of 1 min at 60‡C, and 1.5 min at 72‡C, and 1 min at 95‡C, using a BioRad iCycler (Bio-Rad, Hercules, CA, USA). Thirty cycles was deemed optimal and was used in subsequent experiments. All PCR products were visualized via gel electrophoresis (1.5% agarose gel) and ethidium bromide staining. Densitometric analysis of the band densities was done with ImageQuanNT software (Molecular Dynamics, Sunnyvale, CA, USA). Sequencing Since two bands were present after gel electrophoresis of the NCX1 PCR product, their respective sequences were determined to ensure that both were ampli¢cations of NCX1 cDNA. Once the bands were su⁄ciently separated by electrophoresis, both products were isolated following the QIAquick Gel Extraction Kit Protocol using a vacuum manifold (Qiagen, Valencia, CA, USA). In brief, each band was excised from the gel and dissolved in QG bu¡er (300 Wl/100 mg of gel). Isopropanol (100 Wl/100 mg of gel) was then added, and the mixture was placed in the QIAquick column with vacuum. The column was rinsed ¢rst with 0.5 ml of QG bu¡er and then 0.75 ml of PE bu¡er. Subsequently, 50 Wl of H2 O was added to the column and centrifuged at 10 000Ug. The water sample, containing the puri¢ed PCR product, was sent to the W.M. Keck Foundation Biotechnology Resource Lab (Yale University School of Medicine) for sequencing. Using dideoxynucleotide chain termination reaction and automated detection (ABI Prism Model 3700; Applied Biosystems, Applera Corporation, Foster City, CA, USA), the precise sequences for both cDNA segments were determined. Western blotting Protein concentrations of each sample were determined using a DC Protein Assay Kit (Bio-Rad). 25 Wg protein samples in Tris-bu¡ered loading solution (2% sodium dodecyl sulfate, 100 mM dithiothreitol, 10% glycerol, 0.02% Bromophenol Blue, and
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20 mM Tris, pH 6.8) were electrophoresed in 10% polyacrylamide gels and transferred to polyvinylidene £uoride membranes (Immobilon-P, Millipore, Bedford, MA, USA) at 350 mA for 3 h at 4‡C. The transfer of protein to the membranes was assessed by Ponceau S staining. Membranes were blocked in Tris-bu¡ered saline with Tween 20 (TTBS) bu¡er (0.1% Tween 20 (Bio-Rad); 20 mM Tris, 500 mM NaCl) with 5% non-fat dry milk (Carnation, Nestle Food Co., Glendale, CA, USA) for 1 h, followed by three rinse cycles in TTBS. Membranes were then incubated with the rabbit anti-NCX1 antibody (1:1000; RBI, Natick, MA, USA and SWANT, Switzerland) in TTBS and 2.5% milk for 3 h. After three rinses in TTBS, membranes were incubated with secondary antibody, anti-rabbit immunoglobulin G (IgG) linked to horseradish peroxidase (1:1000; Zymed, South San Francisco, CA, USA) in TTBS and 2.5% milk for 1 h. Signal detection was carried out using the ECL chemiluminescence system (Amersham, Little Chalfont, UK). All membranes were stripped, and re-probed with a mouse
67
monoclonal antibody to L-actin (Chemicon, Temecula, CA, USA). Densitometric analysis of the immunoblot ¢lms was conducted on a Personal Densitometer SI scanner and ImageQuaNT analysis software (Molecular Dynamics). Data analysis mRNA measurements were ¢rst normalized to GAPDH and then expressed as a percentage of the respective P23 group, while NCX1 protein measurements were normalized to total protein and expressed as a percentage of the respective P23 density located on the same membrane. This P23 group was chosen rather than the P30 group due to the limited expression observed in NCX1 mRNA at P30. All data are represented as mean X S.E.M. and were subjected to statistical analysis with the Student’s t-test. Data analysis was performed using GraphPad Prism 3.0 software (GraphPad Software, Inc., San Diego, CA, USA).
Fig. 1. Speci¢city of RT-PCR ampli¢cation. (A) Gel electrophoresis of the NCX1 and NCX2 RT-PCR products with a 100 bp DNA ladder is shown. NCX1 and NCX2 products were ampli¢ed from the same RT samples and electrophoresed through a 1.5% agarose gel. The predicted sizes for the NCX1 ampli¢cation products were 693 bp and 627 bp, while ampli¢cation of the NCX2 isoform resulted in a single 501 bp product. (B) cDNA sequence comparison of the two NCX1 bands is shown. After sequencing, the NCX1 bands were identi¢ed as identical sequences with the exception of a 66 bp omission in the lower band, indicated by the non-shaded area. Further analysis identi¢ed them as splice variants of the NCX1 gene, yielding the notation NCX1-V1 and NCX1-V2.
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G. T. Gibney et al. RESULTS
Speci¢city of NCX primers and isoform identi¢cation Two sets of primers were used for cDNA ampli¢cation of NCX1 and NCX2 in the rat cortex. The primers chosen for NCX1 were speci¢cally designed to amplify the variable region of the NCX1 mRNA transcript where alternative splicing occurs. This allowed quanti¢cation of both total NCX1 mRNA and individual NCX1 splice variants. Gel electrophoresis revealed the presence of two di¡erent PCR products with NCX1 primers, but only a single band from NCX2 ampli¢cation (Fig. 1A). Subsequent sequencing of the NCX1 products showed that the two transcripts shared 97^98% sequence similarity with the previously reported rat brain NCX1 sequence (Furman et al., 1993). As represented in Fig. 1B, the smaller NCX1 band, designated NCX1-V2, differs from the larger NCX1 band, designated NCX1-V1, by a 66 bp deletion from bases 2049^2116. Also of signi¢cance is that the predicted sizes of the NCX1 and NCX2 bands correspond well to the observed sizes determined from gel electrophoresis with the 100 bp DNA marker (Fig. 1A). RT dilution and cycle optimization In order to determine the optimal conditions for the polymerase chain reaction assays, various RT product volumes and cycle numbers were utilized for samples at P1 and P23. These ages were chosen since our preliminary data revealed that signi¢cant developmental di¡erences in expression existed for NCX and it would be necessary to incorporate this factor into our experimental design. As shown in Fig. 2A, three RT volumes (2.5, 5 and 10 Wl), as well as a negative control, were assayed. The 10 Wl lanes appear to approach signal saturation when compared to the 5 Wl lanes, especially in NCX2. Since the band density at 2.5 Wl was either faint or not present, a volume of 5 Wl for the RT product was optimal. Also, it should be noted that no bands were present in negative controls (0 Wl lanes), suggesting that our experiments were not contaminated by any source of exogenous RNA. In terms of cycle optimization (Fig. 2B), 30 cycles yielded the best ampli¢cation product for NCX1, NCX2 and GAPDH. After 35 cycles, NCX2 band density was over-saturated and demonstrated some non-speci¢c ampli¢cation, while GAPDH intensity remained constant or slightly decreased in comparison to 30 cycles. With only 25 cycles, no bands or only faint bands were observed in each group. Based on these experiments, it was determined that an RT volume of 5 Wl and a cycle number of 30 were optimal for measuring the developmental expression of NCX. Expression of NCX1 mRNA in the developing cortex The expression of NCX1 mRNA (NCX1-V1 and NCX1-V2) in the developing cortex was determined at six di¡erent ages, i.e. E22, P4, P8, P13, P23, and P30. RT-PCR was conducted three times for each RNA sam-
Fig. 2. Optimization experiments for RT-PCR assays. (A) Gel electrophoresis of NCX1 and NCX2 RT-PCR products for di¡erent RT volumes (RT: 0, 2.5, 5 and 10 Wl) is shown. A volume of 0 Wl RT product was used as a negative control. In both NCX1 and NCX2 assays, 2.5 Wl of RT product yielded weak band signals after PCR ampli¢cation, whereas band densities from 10 Wl volumes were largely over-saturated. (B) Gel electrophoresis of NCX1, NCX2 and GAPDH RT-PCR products from di¡erent cycles (25 cycles: C25; 30 cycles: C30; 35 cycles : C35) is shown. The band densities from 25 cycles of PCR ampli¢cation were weak or below detection levels, while band densities from 35 cycles were over-saturated. Note that P1 and P23 age groups were assessed for optimal conditions since NCX expression varies substantially during development.
ple, and the individual values from each assay were averaged together to ensure accurate assessment. Each age group from di¡erent six cortex samples represents the average NCX1 mRNA density. Total NCX1 mRNA expression was relatively low in the youngest age group and rose dramatically to a peak level at P8 (Fig. 3A, B). Beyond this age, NCX1 mRNA levels steadily decreased, reaching a minimal density level at P30. The validity of these data was supported by the relatively constant expression levels of GAPDH mRNA throughout development. In addition, the NCX1 splice variants were differentially expressed (Fig. 3C). Both NCX1-V1 and NCX1-V2 were initially present in approximately equal amounts. With increasing age, the proportion of NCX1V1 mRNA steadily increased, while that of NCX1-V2 steadily decreased. At P30 and thereafter, NCX1-V2 levels reached undetectable levels, leaving only NCX1-V1 present at this age.
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was applied as was conducted for NCX1 measurements. The results showed a pattern of expression that was dissimilar to that of NCX1 mRNA expression (Fig. 4). Initially, the relative expression of NCX2 mRNA was low during early development (embryo to neonate). This level substantially increased by P8, reaching a plateau thereafter. Furthermore, di¡erences in expression levels between NCX1 and NCX2 at various ages could be analyzed, since both NCX isoforms were individually ampli¢ed from the same RT products. The expression level of NCX2 mRNA was found to be 2 to 4.5-fold greater than the level of total NCX1 mRNA present in each age group, even though the band size of NCX2 (501 bp) was smaller than either NCX1 band sizes (693 and 627 bp). For example, the average density of total NCX1 normalized to GAPDH at P8 was 0.162 units, whereas that of NCX2 was 0.466 units, a three-fold di¡erence in expression levels. NCX1 protein expression in the cortex and other brain regions during development To determine the developmental expression of NCX1 protein, western blot assays were performed using a speci¢c anti-NCX1 antibody (Fig. 5), which crossreacts with
Fig. 3. NCX1 mRNA expression during development of the cortex. (A) Gel electrophoresis of the NCX1 RT-PCR products at ages P4, P8, P13 and P23 is shown. (B) The developmental pro¢le of total NCX1 mRNA expression for ages E22 through P30 is shown. Densitometric measurements of the NCX1 RT-PCR products were normalized to GAPDH and then calculated as a percentage of their respective P23 groups. Each group is represented as the mean X S.E.M. of six individual samples. P values: P = 0.01, P4 vs P23, P8 vs P23, P13 vs P30; P 6 0.001, P4 vs P30, P8 vs P30, P23 vs P30. The /embryonic group was not compared to other groups since the data were obtained from 2 pooled samples (6^8 tissues per pool). Note that NCX1 exhibits low expression during early development, reaches maximal levels at P8 and declines thereafter. (C) The di¡erential expression of NCX1 splice variants for each age group is shown. Data are represented as the mean X S.E.M. of the percentage of total NCX1 product density for each age group. The asterisk (D) represents a statistical di¡erence (P 6 0.05) from NCX1-V1 density based on Student’s t-test analysis. Note that NCX1-V1 is the predominant variant expressed with further development of the cortex. Also, NCX1-V2 density in P30 samples was below detectable levels.
Expression of NCX2 mRNA in the developing cortex To study the developmental expression of NCX2 mRNA, PCR was carried out utilizing speci¢c NCX2 primers to amplify the reverse transcript of each sample. The same approach for accurate RT-PCR assessment
Fig. 4. NCX2 mRNA expression during development of the cortex. (A) Gel electrophoresis of the NCX2 RT-PCR product at ages P4, P8, P13 and P23 is shown. (B) The developmental pro¢le of NCX2 mRNA expression for ages E22 through P30 is shown. Densitometric measurements of the NCX2 RT-PCR products were normalized to GAPDH and then calculated as a percentage of their respective P23 groups. Each group is represented as the mean X S.E.M. of six individual samples. P values: P 6 0.05, P4 vs P30; P 6 0.0001, P4 vs P8, P4 vs P13, P4 vs P23. The embryonic group was not subject to statistical comparison because the data were obtained from 2 pooled samples (6^8 tissues per pool). Note that NCX2 exhibits low expression in early ages, and increases to adult levels by P8.
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both NCX1 variants. Since NCX1 mRNA decreased substantially after P8^13, we extended our NCX1 protein measurements to P105 to determine if there was a subsequent decline in expression with further maturation. Also, P8 group was omitted since only slight changes existed in mRNA densities between successive ages. In the cortex, NCX1 protein levels were low in embryonic tissue (E16^18: 40% of P23) and rose signi¢cantly to a peak level by P13 (Fig. 5A, B). After P13, NCX1 protein density declined, yielding a trend that parallels the mRNA results. With further maturation from P30 to P105, the level of NCX1 protein continued to decline. To determine whether the developmental pattern of NCX1 protein is cortex-speci¢c, we further compared
NCX1 protein expression in the cortex to that in the brainstem/diencephalon and cerebellum. As was observed in the developing cortex, NCX1 protein expression in the subcortex was lowest in the embryonic age group (E16^18: 35% of P23) and increased steadily through P13 (110% of P23) (Fig. 5C, D). With age, NCX1 expression decreased to adult levels. After 105 days of postnatal growth, NCX1 protein expression decreased to levels comparable to those of newborn NCX1 densities (P105: 65% of P23). In contrast, NCX1 protein expression in the cerebellum did not statistically change during development, although there was a tendency to decrease with age (Fig. 5E, F). From newborn to P23, NCX1 density remained constant with only
Fig. 5. Developmental pro¢le of NCX1 protein expression. Western blots of NCX1 at ages E16, P1, P13, P23, P30 and P105 are shown for the cortical (A), subcortical (C) and cerebellar (E) regions. 25 Wg of total protein was loaded onto 10% polyacrylamide gels, separated by sodium dodecyl sulfate^polyacrylamide gel electrophoresis for 1 h at 100 mV and transferred to polyvinylidene £uoride membranes. Membranes were probed with a highly speci¢c NCX1 antibody. Densitometric measurements of NCX1 band densities at ages E16 through P105 are shown for cortical (B), subcortical (D) and cerebellar (F) regions. Measurements were normalized to total membrane protein and expressed as a percentage of the P23 group. Each group is represented as mean values X S.E.M. from three samples. P values in B (cortex): P 6 0.05, E vs P13, E vs P23, P13 vs P105, P23 vs P105; P = 0.001, P13 vs P30, P23 vs P30; P 6 0.0001, P13 vs P23. P values in D (subcortex) : P 6 0.05, E vs P1, E vs P13, E vs P30, P1 vs P13, P13 vs P23, P30 vs P105; P = 0.005, P1 vs P105; P 6 0.001, E vs P23, P1 vs P23, P13 vs P105; P 6 0.0001, P23 vs P105.
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slight deviations (P1: 102% of P23; P13: 90% of P23). Beyond this stage of development, NCX1 expression tended to decline (P30: 95% of P23; P105: 70% of P23). Note that cerebellar NCX1 protein density was not measured in the E16^18 age group because the limited size of the cerebellum was not su⁄cient for protein assessment.
DISCUSSION
In the present study we have made several important observations pertaining to the NCX in the developing rat cortex: (1) NCX1 splice variant mRNA is di¡erentially expressed depending on age; (2) NCX1 and NCX2 mRNA expression increases from fetus to P8, but diverges thereafter with a substantial decline in NCX1 density and a constant level of NCX2 density; and (3) in spite of its low density in the fetus, NCX1 protein expression remains at a relatively constant level from newborn to adult. Di¡erential splicing of NCX1 during cortical development Within the alternative splicing region of the NCX1 mRNA isoform, there are six identi¢able cassette exons, designated exons A, B, C, D, E and F (Kofuji et al., 1994; Quednau et al., 1997). Although there have been as many as nine di¡erent NCX1 splice variants identi¢ed in adult rat tissues, only three distinct splicing combinations (ACD, AD and ADF) have been observed in the brain (Kofuji et al., 1994; Quednau et al., 1997). In this work, we utilized speci¢c primers to detect all variants and found that only two NCX1 splice variants, NCX1-V1 and NCX1-V2, are present in the developing cortex. NCX1-V1 contained exons ADF and NCX1-V2 contained exons AD, which correspond to the splice variant notation NCX1.5 and NCX1.4 (Quednau et al., 1997), respectively. Interestingly, the exclusion of the short cassette exon F was found to di¡er between ages. With maturation of the cortex, the proportion of NCX1-V1 mRNA in relation to total NCX1 mRNA steadily increased, while the proportion of NCX1-V2 vastly declined, demonstrating that the expression of NCX1 splice variants is age-dependent. The di¡erential regulation of NCX1 splice variant expression may be of signi¢cance in neuronal function. Past investigations have demonstrated that NCX1.4 responds di¡erently to applied physiological conditions as compared to splice variants NCX1.1 and NCX1.3 (Matsuoka et al., 1995; Dyck et al., 1999). NCX1.5 and NCX1.4, the two splice variants observed in this study, have not been subjected to functional comparison. However, the inclusion/exclusion of exon F may yield a functional di¡erence between these variants. So far, multiple sites for possible phosphorylation via protein kinases have been identi¢ed on the cytoplasmic loop (Pearson and Kemp, 1991; Matsuda et al., 1997). This F exon lies adjacent to consensus regulatory domains and contains three serine residues (Quednau et al., 1997) that may be potential sites for phosphorylation.
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Moreover, such regulation has been observed in vascular smooth muscle cells where the phosphorylation of multiple serine residues led to stimulation in NCX activity (Matsuda et al., 1997). If the inclusion of cassette exon F by NCX1.5 o¡ers a regulatory advantage over NCX1.4, neuronal development may selectively favor the expression of the former splice variant as maintenance of calcium homeostasis becomes increasingly important with further maturation (Jiang and Guro¡, 1997; Finkbeiner, 2000). Developmental changes in NCX mRNA and protein Little is known about the developmental expression of NCX subtypes in the cortex and other regions of the brain. Our semi-quantitative PCR data suggest that NCX1 and NCX2 mRNA are di¡erentially expressed in the developing cortex. Since constant GAPDH mRNA band densities were observed throughout the development assays, we are con¢dent that the disparity between NCX mRNA levels represents a speci¢c change in NCX expression during development. In addition to mRNA analysis, NCX1 membrane protein expression was assessed in the developing cortex, as well as diencephalon/brainstem and cerebellar regions. We chose not to normalize the NCX1 protein densities to L-actin levels because in this study, and in other recent work by our group, we have found that L-actin membrane protein density is not constant in various brain regions throughout development (Douglas et al., 2001). This developmental alteration in L-actin expression may be related to either an increase in neuronal populations that occurs in postnatal rat development or to an increase in overall protein density per cell (Douglas et al., 2001). With either event occurring, proteins that are normally constitutively expressed may not serve as stable reference points for extended developmental periods, such as those analyzed in this study. Therefore, NCX1 protein densities were presented to show relative changes between ages, while L-actin protein levels were utilized for the assessment of general protein loading and membrane transfer e⁄cacy of the western blots, as we have done in previous investigations (Douglas et al., 2001). So far it is not clear as to which NCX isoforms signi¢cantly contribute to the increase in Naþ /Ca2þ exchange capacity with maturation of the mammalian brain (Juha¤szova¤ and Rus›e'a¤k, 1991; Sakaue et al., 2000). Our experiments demonstrated that NCX1 and NCX2 mRNA pro¢les diverge after an early increase in expression, with a decline in NCX1 mRNA and constant NCX2 levels beyond P8. Also, NCX2 mRNA expression is signi¢cantly greater than that of NCX1 mRNA in all age groups. This clearly indicates that NCX2 plays a dominant role in Naþ /Ca2þ exchange at all ages. The elevated expression of NCX2 may account for the observed developmental increase in ionic exchange. This is further supported by our NCX1 protein expression data in demonstrating no signi¢cant difference between newborn and adult levels. One of the mechanisms of hypoxic injury via intracel-
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lular Ca2þ overload is believed to involve ‘reverse mode operation’ of NCX (Stys et al., 1991; LoPachin et al., 1997). Indeed, the non-speci¢c NCX inhibitor, KBR7943, has been shown to protect CA1 neurons from hypoxia-induced injury (Schroder et al., 1999). However, it is unclear whether developmental changes in the NCX expression correspond to di¡erences in hypoxic susceptibility between newborn and adult tissues. Our results show that NCX1 mRNA levels are higher in the newborn than in adult and that NCX1 protein expression is approximately equal in the immature and mature brain, including the cortical, subcortical and cerebellar regions. These data suggest that NCX1 expression may not be directly related to age-dependent susceptibility. On the contrary, NCX2 mRNA increased with age and maintained constant levels with further development. Whether its developmental expression is related to the increase in hypoxic susceptibility with age is not clear at present.
In conclusion, our ¢ndings have elucidated the developmental expression patterns of NCX1 and NCX2 in the rat cortex. Our results suggest that NCX2 may play a predominant role in Naþ /Ca2þ exchange in the developing and mature cortex, while NCX1 protein expression does not substantially change between newborn and adult levels. Since NCX has been linked to hypoxia-induced Ca2þ overload (Stys et al., 1991; LoPachin et al., 1997), further study is important to determine whether this developmental increase in NCX2 expression is related to the observed di¡erences in hypoxic susceptibility between immature and mature neurons.
Acknowledgements/This work was supported by March of Dimes (FY00-722) and NIH (R01 HD-34852) to Y.X. and NIH grants (PO1 HD-32573 and RO1 NS-35918) to G.G.H.
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