Developmental age-dependent upregulation of choline acetyltransferase and vesicular acetylcholine transporter mRNA expression in neonatal rat septum by nerve growth factor

ELSEVIER

Neuroscience Letters 209 (1996) 134-136

NBIROtGNNGI LEITERS

Developmental age-dependent upregulation of choline acetyltransferase and vesicular acetylcholine transporter mRNA expression in neonatal rat septum by nerve growth factor Xintian Tian, Xiaoyan Sun, Janusz B. Suszkiw* Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, P.O. Box 670576, 231 Bethesda Avenue. Cincinnati, OH 45267-0576, USA

Received 20 January 1996; revised version received 25 March 1996; accepted 5 April 1996

Abstract

We examined the effect of intraventricular injection of nerve growth factor (NGF) on the choline acetyltransferase (CHAT) and vesicular acetylcholine transporter (VAChT) mRNA expression in the septa of neonatal rats. Rat pups were injected with 2.5 S NGF or cytochrome-c (control) on postnatal days (PN) 4 and 18, and sacrificed 3 days after injections for analysis of ChAT and VAChT mRNA levels by dot-blot hybridization of total septal RNA. In the NGF-treated pups, the ChAT and VAChT mRNA levels were elevated 3- and 2-fold, respectively, at PN7, and 1.8- and 1.3-fold at PN21. These results indicate that (1) NGF upregulates the expression of both ChAT and VAChT genes, (2) NGF has a greater effect on the expression of ChAT mRNA than VAChT mRNA, and (3) the effect of exogenous NGF on the expression of both genes diminishes with developmental age. Keywords: Rat; Septum; Vesicular acetylcholine transporter; Choline acetyltransferase; mRNA; Nerve growth factor

Several lines of evidence indicate that nerve growth factor (NGF) plays an important physiological role in regulating phenotypic development and growth of the cholinergic septohippocampal projection neurons [8]. Administration of NGF to neonate rats in vivo or to septal neurons in culture induces a several-fold increase in the expression of the acetylcholine biosynthetic enzyme choline acetyltransferase (CHAT) activity [10,11,14,15] and ChAT mRNA levels [6,15]. NGF-responsive elements have been mapped to the proximal sequences of the promoter region of the ChAT gene [2,13]. Another cholinergic phenotype-specific protein which is also likely to be regulated by NGF is the vesicular acetylcholine transporter (VAChT) [19]. The rat VAChT gene has been recently cloned [9,18] and shown to reside within the first intron of the ChAT gene [1], suggesting that expression of both genes may be coordinately regulated [1,9,19]. Coordinated upregulation of ChAT and VAChT gene expression by retinoic acid, cAMP, and the leukemia inhibitory factor/ciliary neurotrophic factor (LIF/CNT) have been reported by several laboratories [3, * Corresponding author. Tel.: +1 513 5583039; fax: +1 513 5585738.

4,16]; however, the effect of NGF on V A C h T m R N A expression has not been yet examined. Rat pups were intraventricularly injected with NGF ( 3 0 # g of 2.5 S NGF) or equivalent amount of cytochrome-C (control group) at PN4 and PN18 and sacrificed by decapitation at 3 days postinjections, i.e., at PN7 and PN21. Septa were dissected out on ice-chilled plate and frozen in liquid nitrogen. Total RNA was extracted with RNAzol T M B (Cinna/Biotecx Laboratories International Inc.), denatured in formaldehyde, loaded at three different concentrations onto magna charge nylon membrane (Micron Separations Inc., MSI) in a dot-blot filtration manifold apparatus, and immobilized under UV light (302 nm for 30 s). The ChAT m R N A was detected by hybridization of the dot-blots with a 32p-labeled 43-mer oligoprobe complementary to the 1818-1860 sequence of the ChAT cDNA [5]. The probe has been shown previously [12] and confirmed by us to selectively hybridize with a 4.0 kb ChAT m R N A in Northern blots. VAChT mRNA was detected with a 32P-labeled riboprobe complementary to VAChT cDNA fragment (nt 1790-2881) which was kindly provided to us by Dr. Erickson. The riboprobe has been shown previously to hybridize spe-

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X. Tian et al. / Neuroscience Letters 209 (1996) 134-136

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Fig. 1. Representative ChAT (A) and VAChT (B) hybridization signals in dot-blots of total septal RNA from PN7 and PN21 rat pups 3 days after injections of cytochrome c (control) or NGF. The 'reference' 18S rRNA probe signals were obtained by rehybridization of the same dot-blots. Because different specific activity probes were used for PN7 and PN21 samples, signal densities are not directly comparable between the two age groups. cifically with a single 3.0 kb m R N A species in Northern blots of polyadenylated R N A from cholinergic regions of rat brain but not with noncholinergic tissues [9]. Constitutively expressed cyclophilin m R N A , used as a negative control, was detected with a 32p-labeled c D N A ( 4 4 361 bp) probe [7]. After overnight hybridization with the respective probes at 42°C, membranes were washed at high stringency in DEPC-treated water containing SSPE, SDS and RNase (for V A C h T only). The washed membranes were exposed from 4 to 16 h and hybridization signal intensities quantified using Phosphorlmager SF (Molecular Dynamics). The same membranes were then rehybridized with a 32p-labeled 21 nt 18S-rRNA oligoprobe [17], which was used to normalize the CHAT, VAChT, and cyclophilin hybridization signals. Representative C h A T m R N A (Fig. 1A), V A C h T

m R N A (Fig. 1B) and the corresponding 18S r R N A hybridization signals in dot-blots of total R N A from the septa of PN7 and PN21 pups are shown in Fig. 1. After ascertaining that the signal intensities were linearly related to the amount of total m R N A loaded onto the membrane, the C h A T and V A C h T probe signals were normalized relative to the 18S rRNA hybridization signal. Combined results from N = 4 animals in control and N G F treatment groups are shown in Table 1. N G F treatment significantly increased septal levels of both C h A T m R N A (NGF/Control = 3) and V A C h T - m R N A (NGF/Control = 2) at PN7. The effect of N G F injection diminished in PN21 animals, wherein the NGF/Control ratios for C h A T m R N A and V A C h T m R N A were =1.8 and 1.3, respectively. Indeed, the increase in V A C h T - m R N A levels in NGF-treated animals was not significant, although this

Table 1 Relative levels of ChAT and VAChT mRNAs in the septa from control and NGF-treated rats PN7

ChAT mRNA/18S rRNA x 100 VAChT mRNA/18S rRNA x 100

PN21

Control

NGF-treated

Control

NGF-treated

0.722 ± 0.192 1.312 ± 0.280

2.155 ± 0.208** 2.696 ± 0.471"

!.784 ± 0.253 4.657 ± 0.651

3.168 ± 0.520* 6.052 ± 1.196

The results are expressed as mean + SD. Number of samples in each group is four. Statistical significance of differences between control and NGF groups was analyzed by the Student t-test; *P < 0.05, **P < 0.001.

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x. Tian et al. / Neuroscience Letters 209 (1996) 134-136

may merely reflect the small n u m b e r o f samples tested. The effect of N G F was selective for cholinergic gene transcripts as the levels of cyclophlin m R N A were not significantly different between control and N G F treatment groups. Thus, the cyclophilin m R N A / 1 8 S r R N A ratios measured in septa from PN21 animals were 8.1 _+ 1.4 and 8 , 3 - 1.1 (mean __ SD, n - - 4 ) in control and N G F treatment groups. These results s h o w that C h A T and V A C h T m R N A expression in septal cholinergic neurons is upregulated in vivo by e x o g e n o u s N G F . The N G F - i n d u c e d increase in the steady-state levels of both transcripts is consistent with the notion that expression o f C h A T and V A C h T may be coordinately regulated through c o m m o n enhancer elements in C h A T - V A C h T gene c o m p l e x [19]. H o w e v e r , it should be noted that N G F appears to increase the C h A T transcript levels to a greater extent than the V A C h T m R N A , suggesting that N G F activates the expression of the two genes in a quantitatively differential manner. Differential effects on C h A T / V A C h T m R N A expression have been previously o b s e r v e d also with c A M P , retinoic acid, and cytokines C D F / L I F and C N T F [4,16]. Finally, the effect o f N G F on both C h A T and V A C h T m R N A expression is most p r o n o u n c e d during the first postnatal week and blunted in 3 - w e e k - o l d pups. This d e v e l o p m e n tal age-related d e c r e a s e in responsiveness to e x o g e n o u s N G F could reflect a saturation of the response by end o g e n o u s N G F which reaches nearly adult levels in the septohippocampal system o f PN21 rat (unpublished observation), and/or represent a switch from inductive to cell survival functions of N G F after PN7 [ 11 ].

[5]

[6]

[7]

[8] [9]

[10]

[11]

[12]

[I 3]

W e thank Dr. J. Erickson, Laboratory o f Cell Biology, N I M H / N I H , for the gift o f p c D N A I plasmid containing the 3 ' - n o n - c o d i n g s e q u e n c e ( 1 7 9 0 - 2 8 8 1 ) of rat V A C h T . This work was supported by Grant n u m b e r ES06365 from the National Institute o f E n v i r o n m e n t a l Health Sciences, NIH.

[14]

[I] Bejanin, S., Cervini, R., Mallet, J. and Berrard, S., A unique gene organization for two cholinergic markers, choline acetyltransferase and putative vesicular transporter of acetylcholine, J. Biol. Chem., 269 (1994) 21944-21947. [2] Bcjanin, S., Habert, E., Berrard, S., Edwards, J.-B.D.M., Loeffler, J.-P. and Mallet, J., Promoter elements of the rat choline acetyltransferase gene allowing nerve growth factor inducibility in transfected primary cultured cells, J. Neurochem., 58 (1992) 1580-1583. [3] Bcrrard, S., Varoqui, H., Cervini, R., Israel, M., Mallet, J. and Diebler, M.F., Coregulation of two embedded gene product, choline acetyltransferase and the vesicular acetylcholine transporter, J. Ncurochem., 65 (1995) 939-942. [4] Berse, B. and Blusztajn, J.K., Coordinated upregulation of choline acetyltransferase and vesicular acetylcholine transporter gene expression by the retinoic acid receptor a, cAMP, and leukemia in-

[16]

[15]

[17]

[18]

[19]

hibitory factor/ciliary neurotrophic factor signaling pathways in murine septal cell line, J. Biol. Chem., 270 (1995) 22101-22104. Brice, A., Berrard, S., Raynaud, B., Ansieau, S., Coppola, T., Weber, M.J. and Mallet, J., Complete sequence of a cDNA encoding an active rat choline acetyltransferase: a tool to investigate the plasticity of cholinergic phenotype express, J. Neurosci. Res., 23 (1989) 266-273. Cavicchioli, L., Flanigan, T.P., Dickson, J.G., Vantini, G., Toso, R.D., Fusco, M., Walsh, F.S. and Leon, A., Choline acetyltransferase messenger RNA expression in developing and adult rat brain: regulation by nerve growth factor, Mol. Brain Res., 9 (1991) 319-325. I)anielson, P.E., Forss-Peter, S., Brown , M.A., Calavetta, L., Douglass, J., Milner, R.J. and Sutcliffe, J.G., plBl5: a cDNA clone of the rat mRNA encoding cyclophilin, DNA, 7 (1988) 261-267. Dreyfus, C.F., Effects of nerve growth factor on cholinergic brain neurons, TIPS, 10 (1989) 145-149. Erickson, J.D., Varoqui, H., Schafer, M.K.H., Modi, M.F.D., Diebler, M.-F., Weihe, E., Rand, J., Eiden, L.E., Bonner, T.I. 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. Gnahn, H., Hefti, F., Heumann, R., Schwab, M. and Thoenen, H., NGF-mediated increase in choline-acetyltransferase (CHAT) in the neonatal forebrain: evidence for a physiological role of NGF in the brain? Dev. Brain Res., 9 (1983) 45-52. Hatanaka, H., Tsukui, H. and Nihonmatsu, I., Developmental change in the nerve growth action from induction of choline acetyltransferase to promotion of cell survival in cultured basal forebrain cholinergic neurons from postnatal rats, Dev. Brain Res., 39 (1988) 85-95. Ibanez, C.F., Ernfors, 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. Ibanez, C.F. and Persson, H., Localization of sequences determining cell type specificity and NGF responsiveness in the promoter region of the rat choline acetyttransferase gene, Eur. J. Neurosci., 3 (1991) 1309-1315. Johnston, M.V., Rutkowski, J.L., Wainer, B.H., Long, J.B. and Mobley, W.C., NGF effects on developing forebrain cholinergic neurons are regionally specific, Neurochem. Res., 12 (1987) 985994. Lorenzi, M.V., Knusel, B., Hefti, F. and Strauss, W.L., Nerve growth factor regulation of choline acetyltransferase gene expression in rat embryo basal forebrain cultures, Neurosci. Lett., 140 (1992) 185-188. Misawa, H., Takahashi, R. and Deguchi, T., Coordinate expression of vesicular acetylcholine transporter and choline acetyltransferase in sympathetic superior cervical neurons, NeuroReport, 6 (1995) 965-968. O'Neill, L., Holbrook, N.J., Fargnoli, J. and Lakatta, E.G., Progressive changes from young adult age to senescence in mRNA for rat cardiac myosin heavy chain genes, Cardioscience, 2 (1991) 1-5. 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. Sci. USA, 91 (1994) 10620-10624. Usdin, T.B., Eiden, L.E., Bonner, T.M. and Erickson, J.D., Molecular biology of the vesicular Ach transporter, TINS, 18 (1995) 218-224.