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Differences in the developmental expression of the vesicular acetylcholine transporter and choline acetyltransferase in the rat brain
HEUROSCIENC[ ELSEVIER
Neuroscience Letters212(1996)107-110
LETTERS
Differences in the developmental expression of the vesicular acetylcholine transporter and choline acetyltransferase in the rat brain Thomas Holler a, Brygida Berse a, Jennifer Made Cermak a, Marie-Fran~oise Diebler ~, Jan Krzysztof Blusztajn a,b,* aDepartment of Pathology, Boston University School of Medicine, 85 E. Newton Street, Boston, MA 02118, USA bDepartment of Psychiatry, Boston University School of Medicine, 85 E. Newton Street, Boston, MA 02118, USA eDL'partement de Neurochiraie, Laboratoire de Neurobiologie Cellulaire, CNRS, 91190 GIF-sur-Yvette, Cedex, France Received 29 January 1996; revised version received 13 May 1996; accepted 4 June 1996
Abstract
The neurotransmitter acetylcholine (ACh) is synthesized by the enzyme choline acetyltransferase (CHAT) and then transported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Since the VAChT gene is located within the first intron of the ChAT gene, it is likely that expression of the two genes is coregulated. We compared the developmental expression of VAChT and ChAT mRNA and protein in rat brain. ChAT mRNA and enzyme activity increased by almost 10-fold from embryonic day 19 to adulthood, with the most pronounced increase occurring after birth. In contrast, VAChT mRNA increased by only about 2-fold from late embryonic stages to adult levels. However, VAChT protein followed the developmental pattern of ChAT activity, revealing a large excess of VAChT mRNA over VAChT protein during early stages of development. The results are suggestive of differential mechanisms of ChAT and VAChT regulation during brain development, and of possible translational control of VAChT expression.
Keywords: Rat; Brain; Development; Vesicular acetylcholine transporter; Choline acetyltransferase; Cholinergic; Northern blot; Western blot
Cholinergic neurotransmission requires the expression of proteins that are responsible for the synthesis, storage, and release of the neurotransmitter acetylcholine (ACh). The synthesis of ACh from choline and acetyl coenzyme A occurs in the cytoplasm of cholinergic neurons and is catalyzed by the enzyme choline acetyltransferase (CHAT). Since ChAT is regarded a specific enzyme for the cholinergic phenotype, determinations of ChAT activity and ChAT mRNA have been widely used as markers for cholinergic neurons and cholinergic function of the brain under both physiological and pathological conditions. Another marker of the cholinergic phenotype is the protein responsible for the storage of ACh into vesicles in cholinergic nerve terminals, the vesicular ACh transporter (VAChT). VAChT belongs to the family of amine transporters and acts as a proton antiporter that exchanges intravesicular protons for its cationic substrate * Corresponding author. Room MI009. Tel.: +1 617 6384829; fax: +1 617 6385400.
ACh, a function which is inhibited by vesamicol [17]. Recently, the genes for human and rat VAChT were cloned and shown to be expressed in rat basal forebrain, basal ganglia, and brainstem, i.e. regions that contain ChAT mRNA expressing cell bodies [19,20]. Interestingly, the VAChT gene is not interrupted by introns and is entirely contained within the first intron of the ChAT gene [1,7]. This unique organization of two genes that are responsible for the expression of the same neurotransmitter phenotype suggested that these genes might be coregulated. Indeed, retinoic acid and leukemia inhibitory factor, stimuli known to enhance the cholinergic phenotype in cholinergic cells or to act as cholinergic differentiation factors, cause an up-regulation of both ChAT and VAChT mRNA in cultured sympathetic neurons [2,16] and in a murine septal cell line [3]. In cultured sympathetic neurons, the increase in VAChT mRNA also increased the number of VAChT molecules as assessed with the use of an anti-VAChT antibody and binding studies with vesamicol [2]. These in vitro studies suggest that
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T. Holler et al./Neuroscience Letters 212 (1996) 107-110
ChAT and VAChT expression may be coregulated in vivo during brain development. The expression of ChAT activity and mRNA during brain development has been extensively studied and used as an indicator for the development of the cholinergic system in the brain [23]. In the present study, we examined the expression of VAChT mRNA and protein levels in rat brain and found considerable differences between VAChT and ChAT expression during prenatal and postnatal development. Brains were obtained from Sprague-Dawley rats on embryonic days (E) 19 and 21 and on postnatal days (P) 1, 3, 7, 14, 24, 34, and 49. Brains from two (on P49) to 10 (at embryonic stages) rats per age-group (50% males and 50% females for each age-group) were removed, pooled and immediately frozen in liquid nitrogen. Pooled brains of each age group were ground together in a mortar in liquid nitrogen and the powders were kept frozen at -80°C until analysis. Total RNA was extracted from the ground brains using the guanidinium thiocyanatephenol/chloroform method [5]. Northern analysis was performed as previously described [3,18]. Briefly, RNA preparations from different stages of development were size-fractionated (25/~g RNA per lane) and transferred to Biotrans membrane (ICN). The blots were hybridized with a 0.87 kb mouse ChAT cDNA probe that corresponds to the 3' end of the coding region [18], or a 0.814 kb mouse cDNA fragment of the VAChT coding region [3]. The hybridizing bands were visualized with Phosphorlmager 400E (Molecular Dynamics) and their intensity was quantified using ImageQuant NT software (Molecular Dynamics). The size of hybridizing mRNAs was estimated based on their positions relative to 28S and 18S rRNAs. For analysis of ChAT activity [8] aliquots of the frozen ground brains were homogenized by sonication in sucrose buffer (0.25 M sucrose, 10 mM HEPES, 1 mM EDTA; pH 7.2). For Western blot analysis of VAChT protein, aliquots of frozen tissue were suspended in lysis buffer (25 mM Tris (pH 7.5), 250 mM NaCI, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 2 m M 4-(2aminoethyl)benzenesulfonyl fluoride, 1/~g/ml leupeptin, 2/~g/ml aprotinin), vortexed vigorously, incubated on ice for 15 min, and cleared by centrifugation. Protein content was determined using the bicinchoninic acid assay with bovine serum albumin as standard [21 ]. The brain extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12%) (75/tg protein per lane) and transferred to Immobilon P membrane (Milli pore). The membrane was blocked in phosphate-buffered saline (PBS) containing 5% non-fat dry milk and 0.5% Tween 20 for 1 h and then probed overnight with a rabbit polyclonal antibody raised against the C-terminus of rat VAChT [2]. The specificity of the antibody was tested in CV1 cells transfected with recombinant rat VAChT cDNA. Western blot analysis of transfected cells showed immunoreactivity to the antibody with the largest band being 64kDa whereas mock-transfected CV1 cells
showed no immunoreactivity to the antibody (data not shown). The size of immunoreactive proteins in rat brain extracts was estimated using Kaleidoscope prestained standards (Bio-Rad). ChAT mRNA (Fig. 1A) detected as a single band of about 4.5 kb, was present at low levels on El9, the earliest age tested. It increased slowly until P7, and rapidly after P7, until it reached peak levels at P24. Quantification of the Northern blots (Fig. 1B) showed an increase of about 6-fold in the abundance of ChAT mRNA between El9 and P24. After P24 no significant changes in ChAT mRNA levels were detected. ChAT activity (Fig. 1B) in brain homogenates was measured as an indicator of ChAT protein levels. At embryonic stages ChAT activity was very low (ACh formation on El9, 2.03 nmol/mg protein per 30 min) and increased by about 25-fold until maximal levels were reached on P49 (52.20 nmol/mg protein per 30 min). Expression of the data for both ChAT mRNA and ChAT activity as % of the maximal values that occurred during development revealed similar developmental patterns with a 2-4 day delay in the in-
An
ChAT mRNA
E19 E21
PI
PC3
P7 P14 P24 P34 P49
Bm 120
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100
60 40 20
=
ChATac~vi~
'1''''1''''1''''1''''1''''1 0 10 20 30 age (days)
40
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Fig. 1. Development of ChAT mRNA and ChAT activity in rat brain. (A) RNA preparations, extracted from rat brains at the indicated stages of development, were hybridized with a mouse ChAT cDNA probe as described in the text. (B) The Northern blots were quantified and RNA levels were expressed as % of the maximal level that occurred on P24. ChAT activity in brain homogenates (50/~g of protein) was measured by the radioenzymatic assay of Fonnum [8] and expressed as % of the maximal value which was measured on P49 (ACh formation: 52.20 ± 0.06 nmol/mg protein per 30 min). 0 days on the abscissa corresponds to the day of birth. Data are presented as means ± range of two experiments for ChAT activity and for mRNA.
T. Holler et al. / Neuroscience Letters 212 (1996) 107-110
creases of ChAT activity relative to those of mRNA (Fig. 1B). VAChT mRNA was detected as a broad band of about 3 kb. The developmental pattern of VAChT mRNA (Fig. 2A) differed from that of ChAT mRNA. Quantification of Northern blots (Fig. 2C) showed that, on E21, mRNA levels for VAChT had already reached 60% of the maximal level which occurred on P24. The most rapid increase of VAChT mRNA was detected between El9 and P3.
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Fig. 2. Development of VAChT mRNA and VAChT protein levels in rat brain. (A) Brain RNA preparations from different stages of development were subjected to Northern blot analysis with a mouse VAChT probe. RNA from rat liver served as a negative control. (B) Brain extracts were analyzed by Western blotting with a polyclonal antiVAChT antibody. The antibody/antigen complexes were detected with goat anti-rabbit lgG pernxidase conjugates, and visualized using the chemiluminescence method (Renaissance reagent, DuPont NEN) and Kodak X-Omat AR film. (C) Northern blots were quantified as in Fig. 1, and Western blots were quantified from the autoradiographic film image with a densitometric scanner and ImageQuant software. The graph shows VAChT mRNA and VAChT protein levels expressed as % of the maximal levels measured. 0 days on the abscissa corresponds to the day of birth. Presented are means ± SEM of four experiments for mRNA and three experiments for protein.
109
After P3 the developmental curve showed only a slow increase. Consistent with the results of Berrard et al. [2] and of Gilmor et al. [9], VAChT protein was detected as a band of 64-67 kDa using Western blot analysis with the polyclonal antibody against rat VAChT. VAChT protein levels increased with a marked delay of about 10-20 days after VAChT mRNA levels, showing a developmental pattern similar to that of ChAT activity (Fig. 2B,C). The present study is the first to examine the developmental pattern of VAChT expression in rat brain on both mRNA and protein levels. VAChT mRNA levels already reached up to 60% of adult levels at late embryonic stages and showed only an about 30% increase after birth. In contrast, ChAT mRNA levels underwent the most pronounced changes postnatally, suggesting that, during development, these genes are regulated differentially. This finding is surprising because, in previous studies that examined the induction of ChAT and VAChT by soluble factors in different cell systems, both genes were regulated in a coordinated fashion [2,3,16]. Moreover, the tissue distribution of ChAT and VAChT mRNAs in the adult brain is virtually identical. However, subtle differences were found in the regulation of expression of both genes, e.g. cAMP increased VAChT mRNA levels more efficiently than ChAT mRNA levels in the murine septal cell line SN56 [3]. Analysis of the ChAT/VAChT gene revealed the presence of several promoters leading to the generation of multiple RNA species which may play a role in the differences in VAChT and ChAT expression during development. For ChAT three different promoters referred to as R, M and N were identified [11,15]. Only those ChAT mRNA species which are transcribed from the R promoter share the 5'-end with a VAChT mRNA species, whereas M and N promoters are located downstream from the VAChT gene [22]. Recently, two specific promoters for VAChT have been identified within the first intron of the ChAT gene from which two VAChT mRNA species are transcribed [4]. Thus, it appears that the closely linked organization of the genes for ChAT and VAChT together with the presence of specific promoter regions allows both coordinated transcription as well as differential regulation. However, the question remains if the differences in the developmental patterns of VAChT and ChAT mRNA are due to differences in transcription rate or mRNA stability. In agreement with the findings of others [6,10,13] we found that the levels of ChAT activity reflected developmental changes in ChAT mRNA levels, with the increase in mRNA preceding that of enzyme activity by about 2 days. This finding indicates an important role for transcriptional regulation of ChAT expression during development. In contrast, the developmental pattern of VAChT protein was different from that of VAChT mRNA, but very similar to the development of ChAT activity, with very low levels prior to P7. Importantly, the development
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T. Holler et al. / Neuroscience Letters 212 (1996) 107-110
of VAChT protein also closely corresponds to that of the vesicular fraction of ACh in rat brain [12]. The comparison of the developmental patterns of VAChT mRNA and protein revealed a large excess of mRNA from late embryonic stages up to early postnatal ages which suggests a role for translational control of VAChT expression. In early stages of development, translation of VAChT mRNA seems to be repressed. It is also possible, however, that VAChT is translated during early development, but that the newly synthesized VAChT protein is rapidly degraded. It remains to be determined if the distribution of VAChT mRNA in the immature brain is restricted to cells that are cholinergic (i.e. that also express CHAT, albeit at low levels) as has been shown for the adult brain [7,19,20]. If this is the case, VAChT mRNA might be a more useful marker to examine the development of the cholinergic system in the brain than ChAT mRNA because of the early appearance of VAChT mRNA at high abundance. Recently, a regulatory region in the rat ChAT gene located upstream of the R promoter has been shown to direct transcription of a reporter gene to cholinergic tissues in transgenic mice [14]. The developmental regulation of the transgene in the spinal cord of the mice paralleled that of endogenous ChAT mRNA. These findings indicate that this regulatory region is important for the specific expression of ChAT in cholinergic tissues. It will be interesting to determine if the same region is involved in regulating cholinergic-specific expression of the VAChT gene and, furthermore, if it is involved in regulating the differential expression of ChAT and VAChT mRNA during development. This work was supported by Grant AG09525 from NIA, National Institutes of Health. [1] Bejanin, S., Cervini, R., Mallet, J. and Berrard, S., A unique gene organization for two cholinergic markers, choline acetyltransferase and a putative vesicular transporter of acetylcholine, J. Biol. Chem., 269 (1994) 21944-21947. [2] Berrard, S., Varoqui, H., Cervini, R., Israel, M., Mallet, J. and Diebler, M.F., Coregulation of two embedded gene products, choline acetyltransferase and the vesicular acetylcholine transporter, J. Neurochem., 65 (1995) 939-942. [3] Berse, B. and Blusztajn, J.K., Coordinated up-regulation of choline acetyltransferase and vesicular acetylcholine transporter gene expression by the retinoic acid receptor a, cAMP, and leukemia inhibitory factor/ciliary neurotrophic factor signaling pathways in a murine septal cell line, J. Biol. Chem., 270 (1995) 2210122104. [4] Cervini, R., Houhon, L., Pradat, P.F., Bejanin, S., Mallet, J. and Berrard, S., Specific vesicular acetylcholine transporter promoters lie within the first intron of the rat choline acetyltransferase gene, J. Biol. Chem., 270 (1995) 24654-24657. [5] Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. [6] Coyle, .I.T. and Yamamura, H.I., Neurochemical aspects of the ontogeuesis of cholinergic neurons in the rat brain, Brain Res., 118 (1976) 429--440.
[7] Erickson, J.D., Varoqui, H., Schiifer, M.K.H., Modi, W., Diebler, M.F., Weihe, E., Rand, J., Eiden, L.E., Bonner, TT and Usdin, T.B., Functional identification of a vesicular acetylcholine transporter and its expression from a 'cholinergic' gene locus, J. Biol. Chem., 269 (1994) 21929-21932. [8] Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. [9] Gilmor, M.L., Nash, N.R., Roghani, A., Edwards, R.H., Yi, H., Hersch, S.M. and Levey, A.I., Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles. J. Neurosci., 16 (1996) 2179-2190. [10] Ibfifiez, C.F., Erofors, P. and Persson, H., Developmental and regional expression of choline acetyltransferase mRNA in the rat central nervous system, J. Neurosci. Res., 29 (1991) 163-171. [11] Kengaku, M., Misawa, H. and Deguchi, T., Multiple mRNA species of choline acetyltransferase from rat spinal cord, Mol. Brain Res., lg (1993) 71-76. [12] Kewitz, H., Pleul, O. and Mann, E,, Pre- and postnatal development and drug induced alt~'atiom of free and bound acetylcholine in rat brain, Naunyn-Schmiedelmrg's Arch. Pharmacol., 298 (1977) 149-155. [13] Ladinsky, H., Consolo, S., Peal, G. and Garattini, S., Acetylcholine, choline and choline acetyltransferase activity in the developing brain of normal and hypothyroid rats, J. Neurochem., 19 (1972) 1947-1952. [14] Ltnnerberg, P., Lendahl, U., Funakoshi, H., Ahdund-Richter, L., Persson, H. and lbfifiez, C.F., Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice, Proc. Natl. Acad. Sci. USA, 92 (1995) 4046---4050. [15] Misawa, H., lshii, K. and Deguchi, T., Gene expression of mouse choline acetyltransferase. Alternative splicing and identification of a highly active promoter region, J. Biol. Chem., 267 (1992) 20392-20399. [16] Misawa, H., Takahashi, R. and Deguchi, R., Coordinate expression of vesicular acetylcholine transporter and choline acetyltransferase in sympathetic superior cervical neurones, NeuroReport, 6 (1995) 965-968. [17] Parsons, SM., Prior, C. and Marshall, l.G., Acetylcholine transport, storage, and release, Int. Rev. Neurobiol., 35 0993) 279390. [18] Pedersen, W.A., Berse, B., Schiller, U., Wainer, B.H. and Blusztajn, J.K., All-trans- and 9-cis-retinoic acid enhance the cholinergic properties of a mudne septal cell line: evidence that the effects are mediated by activation of retinoic acid receptor-a, J. Neurochem., 65 (1995) 50-58. [19] Roghani, A., Feldman, J., Kohan, S.A., Shirzadi, A., Gundersen, C.B., Brecha, N. and Edwards, R.H., Molecular cloning of a putative vesicular transporter for acetylcholine, Proc. Natl. Acad. USA, 91 (1994) 10620-10624. [20] Sch~ifer, M.K.H., Weihe, E., Varoqui, H., Eiden, L.E. and Erickson, J.D., Distribution of the vesicular acetylcholine transporter (VAChT) in the central and peripheral nervous system of the rat, J. Mol. Neurosci., 5 (1994) 1-26. [21] Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., OIson, B.J. and Klenk, D.C., Measurement of protein using bicinchoninic acid, Anal. Biochem., 150 (1985) 76-85 (published erratum appears in Anal. Biochem., 163 0987) 279). [22] Usdin, T.B., Eiden, L.E., Bonner, T.l. and Erickson, J.D., Molecular biology of the vesicular ACh transporter, Trends Neurosci., 18 (1995) 218-224. [23] Wu, D. and Hersh, L.B., Choline acetyltransferase: celebrating its fiftieth year, J. Neurochem., 62 (1994) 1653-1663.
Neuroscience Letters212(1996)107-110
LETTERS
Differences in the developmental expression of the vesicular acetylcholine transporter and choline acetyltransferase in the rat brain Thomas Holler a, Brygida Berse a, Jennifer Made Cermak a, Marie-Fran~oise Diebler ~, Jan Krzysztof Blusztajn a,b,* aDepartment of Pathology, Boston University School of Medicine, 85 E. Newton Street, Boston, MA 02118, USA bDepartment of Psychiatry, Boston University School of Medicine, 85 E. Newton Street, Boston, MA 02118, USA eDL'partement de Neurochiraie, Laboratoire de Neurobiologie Cellulaire, CNRS, 91190 GIF-sur-Yvette, Cedex, France Received 29 January 1996; revised version received 13 May 1996; accepted 4 June 1996
Abstract
The neurotransmitter acetylcholine (ACh) is synthesized by the enzyme choline acetyltransferase (CHAT) and then transported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Since the VAChT gene is located within the first intron of the ChAT gene, it is likely that expression of the two genes is coregulated. We compared the developmental expression of VAChT and ChAT mRNA and protein in rat brain. ChAT mRNA and enzyme activity increased by almost 10-fold from embryonic day 19 to adulthood, with the most pronounced increase occurring after birth. In contrast, VAChT mRNA increased by only about 2-fold from late embryonic stages to adult levels. However, VAChT protein followed the developmental pattern of ChAT activity, revealing a large excess of VAChT mRNA over VAChT protein during early stages of development. The results are suggestive of differential mechanisms of ChAT and VAChT regulation during brain development, and of possible translational control of VAChT expression.
Keywords: Rat; Brain; Development; Vesicular acetylcholine transporter; Choline acetyltransferase; Cholinergic; Northern blot; Western blot
Cholinergic neurotransmission requires the expression of proteins that are responsible for the synthesis, storage, and release of the neurotransmitter acetylcholine (ACh). The synthesis of ACh from choline and acetyl coenzyme A occurs in the cytoplasm of cholinergic neurons and is catalyzed by the enzyme choline acetyltransferase (CHAT). Since ChAT is regarded a specific enzyme for the cholinergic phenotype, determinations of ChAT activity and ChAT mRNA have been widely used as markers for cholinergic neurons and cholinergic function of the brain under both physiological and pathological conditions. Another marker of the cholinergic phenotype is the protein responsible for the storage of ACh into vesicles in cholinergic nerve terminals, the vesicular ACh transporter (VAChT). VAChT belongs to the family of amine transporters and acts as a proton antiporter that exchanges intravesicular protons for its cationic substrate * Corresponding author. Room MI009. Tel.: +1 617 6384829; fax: +1 617 6385400.
ACh, a function which is inhibited by vesamicol [17]. Recently, the genes for human and rat VAChT were cloned and shown to be expressed in rat basal forebrain, basal ganglia, and brainstem, i.e. regions that contain ChAT mRNA expressing cell bodies [19,20]. Interestingly, the VAChT gene is not interrupted by introns and is entirely contained within the first intron of the ChAT gene [1,7]. This unique organization of two genes that are responsible for the expression of the same neurotransmitter phenotype suggested that these genes might be coregulated. Indeed, retinoic acid and leukemia inhibitory factor, stimuli known to enhance the cholinergic phenotype in cholinergic cells or to act as cholinergic differentiation factors, cause an up-regulation of both ChAT and VAChT mRNA in cultured sympathetic neurons [2,16] and in a murine septal cell line [3]. In cultured sympathetic neurons, the increase in VAChT mRNA also increased the number of VAChT molecules as assessed with the use of an anti-VAChT antibody and binding studies with vesamicol [2]. These in vitro studies suggest that
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ChAT and VAChT expression may be coregulated in vivo during brain development. The expression of ChAT activity and mRNA during brain development has been extensively studied and used as an indicator for the development of the cholinergic system in the brain [23]. In the present study, we examined the expression of VAChT mRNA and protein levels in rat brain and found considerable differences between VAChT and ChAT expression during prenatal and postnatal development. Brains were obtained from Sprague-Dawley rats on embryonic days (E) 19 and 21 and on postnatal days (P) 1, 3, 7, 14, 24, 34, and 49. Brains from two (on P49) to 10 (at embryonic stages) rats per age-group (50% males and 50% females for each age-group) were removed, pooled and immediately frozen in liquid nitrogen. Pooled brains of each age group were ground together in a mortar in liquid nitrogen and the powders were kept frozen at -80°C until analysis. Total RNA was extracted from the ground brains using the guanidinium thiocyanatephenol/chloroform method [5]. Northern analysis was performed as previously described [3,18]. Briefly, RNA preparations from different stages of development were size-fractionated (25/~g RNA per lane) and transferred to Biotrans membrane (ICN). The blots were hybridized with a 0.87 kb mouse ChAT cDNA probe that corresponds to the 3' end of the coding region [18], or a 0.814 kb mouse cDNA fragment of the VAChT coding region [3]. The hybridizing bands were visualized with Phosphorlmager 400E (Molecular Dynamics) and their intensity was quantified using ImageQuant NT software (Molecular Dynamics). The size of hybridizing mRNAs was estimated based on their positions relative to 28S and 18S rRNAs. For analysis of ChAT activity [8] aliquots of the frozen ground brains were homogenized by sonication in sucrose buffer (0.25 M sucrose, 10 mM HEPES, 1 mM EDTA; pH 7.2). For Western blot analysis of VAChT protein, aliquots of frozen tissue were suspended in lysis buffer (25 mM Tris (pH 7.5), 250 mM NaCI, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 2 m M 4-(2aminoethyl)benzenesulfonyl fluoride, 1/~g/ml leupeptin, 2/~g/ml aprotinin), vortexed vigorously, incubated on ice for 15 min, and cleared by centrifugation. Protein content was determined using the bicinchoninic acid assay with bovine serum albumin as standard [21 ]. The brain extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12%) (75/tg protein per lane) and transferred to Immobilon P membrane (Milli pore). The membrane was blocked in phosphate-buffered saline (PBS) containing 5% non-fat dry milk and 0.5% Tween 20 for 1 h and then probed overnight with a rabbit polyclonal antibody raised against the C-terminus of rat VAChT [2]. The specificity of the antibody was tested in CV1 cells transfected with recombinant rat VAChT cDNA. Western blot analysis of transfected cells showed immunoreactivity to the antibody with the largest band being 64kDa whereas mock-transfected CV1 cells
showed no immunoreactivity to the antibody (data not shown). The size of immunoreactive proteins in rat brain extracts was estimated using Kaleidoscope prestained standards (Bio-Rad). ChAT mRNA (Fig. 1A) detected as a single band of about 4.5 kb, was present at low levels on El9, the earliest age tested. It increased slowly until P7, and rapidly after P7, until it reached peak levels at P24. Quantification of the Northern blots (Fig. 1B) showed an increase of about 6-fold in the abundance of ChAT mRNA between El9 and P24. After P24 no significant changes in ChAT mRNA levels were detected. ChAT activity (Fig. 1B) in brain homogenates was measured as an indicator of ChAT protein levels. At embryonic stages ChAT activity was very low (ACh formation on El9, 2.03 nmol/mg protein per 30 min) and increased by about 25-fold until maximal levels were reached on P49 (52.20 nmol/mg protein per 30 min). Expression of the data for both ChAT mRNA and ChAT activity as % of the maximal values that occurred during development revealed similar developmental patterns with a 2-4 day delay in the in-
An
ChAT mRNA
E19 E21
PI
PC3
P7 P14 P24 P34 P49
Bm 120
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60 40 20
=
ChATac~vi~
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Fig. 1. Development of ChAT mRNA and ChAT activity in rat brain. (A) RNA preparations, extracted from rat brains at the indicated stages of development, were hybridized with a mouse ChAT cDNA probe as described in the text. (B) The Northern blots were quantified and RNA levels were expressed as % of the maximal level that occurred on P24. ChAT activity in brain homogenates (50/~g of protein) was measured by the radioenzymatic assay of Fonnum [8] and expressed as % of the maximal value which was measured on P49 (ACh formation: 52.20 ± 0.06 nmol/mg protein per 30 min). 0 days on the abscissa corresponds to the day of birth. Data are presented as means ± range of two experiments for ChAT activity and for mRNA.
T. Holler et al. / Neuroscience Letters 212 (1996) 107-110
creases of ChAT activity relative to those of mRNA (Fig. 1B). VAChT mRNA was detected as a broad band of about 3 kb. The developmental pattern of VAChT mRNA (Fig. 2A) differed from that of ChAT mRNA. Quantification of Northern blots (Fig. 2C) showed that, on E21, mRNA levels for VAChT had already reached 60% of the maximal level which occurred on P24. The most rapid increase of VAChT mRNA was detected between El9 and P3.
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Fig. 2. Development of VAChT mRNA and VAChT protein levels in rat brain. (A) Brain RNA preparations from different stages of development were subjected to Northern blot analysis with a mouse VAChT probe. RNA from rat liver served as a negative control. (B) Brain extracts were analyzed by Western blotting with a polyclonal antiVAChT antibody. The antibody/antigen complexes were detected with goat anti-rabbit lgG pernxidase conjugates, and visualized using the chemiluminescence method (Renaissance reagent, DuPont NEN) and Kodak X-Omat AR film. (C) Northern blots were quantified as in Fig. 1, and Western blots were quantified from the autoradiographic film image with a densitometric scanner and ImageQuant software. The graph shows VAChT mRNA and VAChT protein levels expressed as % of the maximal levels measured. 0 days on the abscissa corresponds to the day of birth. Presented are means ± SEM of four experiments for mRNA and three experiments for protein.
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After P3 the developmental curve showed only a slow increase. Consistent with the results of Berrard et al. [2] and of Gilmor et al. [9], VAChT protein was detected as a band of 64-67 kDa using Western blot analysis with the polyclonal antibody against rat VAChT. VAChT protein levels increased with a marked delay of about 10-20 days after VAChT mRNA levels, showing a developmental pattern similar to that of ChAT activity (Fig. 2B,C). The present study is the first to examine the developmental pattern of VAChT expression in rat brain on both mRNA and protein levels. VAChT mRNA levels already reached up to 60% of adult levels at late embryonic stages and showed only an about 30% increase after birth. In contrast, ChAT mRNA levels underwent the most pronounced changes postnatally, suggesting that, during development, these genes are regulated differentially. This finding is surprising because, in previous studies that examined the induction of ChAT and VAChT by soluble factors in different cell systems, both genes were regulated in a coordinated fashion [2,3,16]. Moreover, the tissue distribution of ChAT and VAChT mRNAs in the adult brain is virtually identical. However, subtle differences were found in the regulation of expression of both genes, e.g. cAMP increased VAChT mRNA levels more efficiently than ChAT mRNA levels in the murine septal cell line SN56 [3]. Analysis of the ChAT/VAChT gene revealed the presence of several promoters leading to the generation of multiple RNA species which may play a role in the differences in VAChT and ChAT expression during development. For ChAT three different promoters referred to as R, M and N were identified [11,15]. Only those ChAT mRNA species which are transcribed from the R promoter share the 5'-end with a VAChT mRNA species, whereas M and N promoters are located downstream from the VAChT gene [22]. Recently, two specific promoters for VAChT have been identified within the first intron of the ChAT gene from which two VAChT mRNA species are transcribed [4]. Thus, it appears that the closely linked organization of the genes for ChAT and VAChT together with the presence of specific promoter regions allows both coordinated transcription as well as differential regulation. However, the question remains if the differences in the developmental patterns of VAChT and ChAT mRNA are due to differences in transcription rate or mRNA stability. In agreement with the findings of others [6,10,13] we found that the levels of ChAT activity reflected developmental changes in ChAT mRNA levels, with the increase in mRNA preceding that of enzyme activity by about 2 days. This finding indicates an important role for transcriptional regulation of ChAT expression during development. In contrast, the developmental pattern of VAChT protein was different from that of VAChT mRNA, but very similar to the development of ChAT activity, with very low levels prior to P7. Importantly, the development
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of VAChT protein also closely corresponds to that of the vesicular fraction of ACh in rat brain [12]. The comparison of the developmental patterns of VAChT mRNA and protein revealed a large excess of mRNA from late embryonic stages up to early postnatal ages which suggests a role for translational control of VAChT expression. In early stages of development, translation of VAChT mRNA seems to be repressed. It is also possible, however, that VAChT is translated during early development, but that the newly synthesized VAChT protein is rapidly degraded. It remains to be determined if the distribution of VAChT mRNA in the immature brain is restricted to cells that are cholinergic (i.e. that also express CHAT, albeit at low levels) as has been shown for the adult brain [7,19,20]. If this is the case, VAChT mRNA might be a more useful marker to examine the development of the cholinergic system in the brain than ChAT mRNA because of the early appearance of VAChT mRNA at high abundance. Recently, a regulatory region in the rat ChAT gene located upstream of the R promoter has been shown to direct transcription of a reporter gene to cholinergic tissues in transgenic mice [14]. The developmental regulation of the transgene in the spinal cord of the mice paralleled that of endogenous ChAT mRNA. These findings indicate that this regulatory region is important for the specific expression of ChAT in cholinergic tissues. It will be interesting to determine if the same region is involved in regulating cholinergic-specific expression of the VAChT gene and, furthermore, if it is involved in regulating the differential expression of ChAT and VAChT mRNA during development. This work was supported by Grant AG09525 from NIA, National Institutes of Health. [1] Bejanin, S., Cervini, R., Mallet, J. and Berrard, S., A unique gene organization for two cholinergic markers, choline acetyltransferase and a putative vesicular transporter of acetylcholine, J. Biol. Chem., 269 (1994) 21944-21947. [2] Berrard, S., Varoqui, H., Cervini, R., Israel, M., Mallet, J. and Diebler, M.F., Coregulation of two embedded gene products, choline acetyltransferase and the vesicular acetylcholine transporter, J. Neurochem., 65 (1995) 939-942. [3] Berse, B. and Blusztajn, J.K., Coordinated up-regulation of choline acetyltransferase and vesicular acetylcholine transporter gene expression by the retinoic acid receptor a, cAMP, and leukemia inhibitory factor/ciliary neurotrophic factor signaling pathways in a murine septal cell line, J. Biol. Chem., 270 (1995) 2210122104. [4] Cervini, R., Houhon, L., Pradat, P.F., Bejanin, S., Mallet, J. and Berrard, S., Specific vesicular acetylcholine transporter promoters lie within the first intron of the rat choline acetyltransferase gene, J. Biol. Chem., 270 (1995) 24654-24657. [5] Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. [6] Coyle, .I.T. and Yamamura, H.I., Neurochemical aspects of the ontogeuesis of cholinergic neurons in the rat brain, Brain Res., 118 (1976) 429--440.
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