Differential expression and regulation of the high-affinity choline transporter CHT1 and choline acetyltransferase in neurons of superior cervical ganglia

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 28 (2005) 303 – 313

Differential expression and regulation of the high-affinity choline transporter CHT1 and choline acetyltransferase in neurons of superior cervical ganglia Marie-Jose´ Lecomte, Ste´phanie De Gois, Aline Guerci, Philippe Ravassard, Nicole Faucon Biguet, Jacques Mallet, and Sylvie Berrard* Laboratoire de la Neurotransmission et des Processus Neurode´ge´ne´ratifs, CNRS, UMR 7091, Baˆtiment CERVI, Hoˆpital de la Pitie´-Salpeˆtrie`re, 83 boulevard de l’Hoˆpital, 75013 Paris, France Received 10 June 2004; revised 17 September 2004; accepted 22 September 2004 Available online 11 November 2004 Previous studies revealed that leukemia inhibitory factor (LIF) and retinoic acid (RA) induce a noradrenergic to cholinergic switch in cultured sympathetic neurons of superior cervical ganglia (SCG) by up-regulating the coordinate expression of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. Here, we examined the effect of both factors on high-affinity choline uptake (HACU) and on expression of the high-affinity choline transporter CHT1. We found that HACU and CHT1-mRNA levels are up-regulated by LIF and down-regulated by RA in these neurons. Thus, in contrast to LIF, RA differentially regulates the expression of the presynaptic cholinergic proteins. Moreover, we showed that untreated SCG neurons express HACU and CHT1-mRNAs at much higher levels than ChAT activity and transcripts. In intact SCG, CHT1-mRNAs are abundant and synthesized by the noradrenergic neurons themselves. This study provides the first example of CHT1 expression in neurons which do not use acetylcholine as neurotransmitter. D 2004 Elsevier Inc. All rights reserved.

Introduction Acetylcholine (ACh) is synthesized in the cytoplasm of cholinergic nerve terminals by choline acetyltransferase (ChAT) from choline and acetyl-coenzyme A. ACh is then translocated by the vesicular ACh transporter (VAChT) into synaptic vesicles, where it is stored until release into the synaptic cleft. Cholinergic signal transduction is terminated by the rapid hydrolysis of ACh into choline and acetate by intrasynaptic acetylcholinesterase. As the capacity of neurons to synthesize choline de novo is limited, choline is supplied from the extracellular fluid by plasma * Corresponding author. Laboratoire de la Neurotransmission et des Processus Neurode´ge´ne´ratifs, CNRS, UMR 7091, Baˆtiment CERVI, Hoˆpital de la Pitie´-Salpeˆtrie`re, 83 boulevard de l’Hoˆpital, 75013 Paris, France. Fax: +33 142177533. E-mail address: [email protected] (S. Berrard). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2004.09.014

membrane transporters. It is well established that the availability of choline in cholinergic cells is the rate-limiting step for ACh synthesis (Jope, 1979; Kuhar and Murrin, 1978; Tucek, 1985). Two kinetically distinct processes for choline uptake have been described for brain synaptosomal preparations (Haga and Noda, 1973; Yamamura and Snyder, 1973): (i) a ubiquitous transport mechanism with a low affinity for choline (Km c 50 AM), by which choline is taken up for the biosynthesis of membrane phospholipids, primarily phosphatidylcholine (PtdCh), and (ii) a high affinity choline uptake (HACU) system (Km = 1–5 AM), by which most of the choline used for ACh synthesis is imported. In contrast to the ubiquitous mechanism, the cholinergic HACU is saturable, Na+and Cl -dependent, and competitively inhibited by low concentrations of hemicholinium-3 (HC-3) with a Ki of 10–100 nM (Guyenet et al., 1973; Haga and Noda, 1973; Jope, 1979; Simon and Kuhar, 1976; Yamamura and Snyder, 1973). HACU is associated with the nerve terminals but not the cell bodies of cholinergic neurons (Bussie`re et al., 2001; Suszkiw et al., 1976), allowing the choline moiety generated by hydrolysis of the ACh released during nerve activity to be recovered. The cholinergic transporter mediating HACU has recently been cloned in several species, including mammals, and is now referred to as CHT1 or CHT (Apparsundaram et al., 2000, 2001; Guermonprez et al., 2002; Okuda and Haga, 2000; Okuda et al., 2000; Wang et al., 2001). Analysis of mutant mice lacking CHT1 confirmed that CHT1 is uniquely required for HC-3-sensitive HACU and ACh synthesis (Ferguson et al., 2004). In the central nervous system, CHT1 colocalizes with ChAT and VAChT, and its expression is restricted exclusively to the cholinergic neurons (Kobayashi et al., 2002; Kus et al., 2003; Lips et al., 2002; Misawa et al., 2001). The determination of the subcellular localization of CHT1 led to the finding that within cholinergic terminals, CHT1 resides mainly in the membrane of synaptic vesicles. These vesicles thus constitute a reservoir from which CHT1 is delivered to the plasma membrane by exocytosis (Ferguson et al., 2003; Nakata et al., 2004; Ribeiro et al., 2003). However, it should be noted that uptake systems with a high affinity

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for choline, which do not share all the properties of CHT1, have also been reported in noncholinergic cells (Lanks et al., 1974; Martin, 1968; Masland and Mills, 1980; Massarelli et al., 1974; Schloss et al., 1994). Thus, a choline transporter cannot be designated as the cholinergic transporter solely on the basis of the criterion of bhigh affinity.Q The discovery of high-affinity choline transporters in noncholinergic cells suggests that several different choline transporters exist, providing various cell types with choline for various uses. This was confirmed by the isolation of a cDNA encoding a protein capable of transporting choline with a high affinity, termed CTL1, which differs from the cholinergic transporter with respect to its ionic dependence and its distribution pattern (O’Regan and Meunier, 2003; O’Regan et al., 2000). Although its physiological function is not clearly understood, CTL1 may be involved in the uptake of choline for membrane synthesis following nerve transection or for myelin lipid synthesis (Che et al., 2002; Meunier and O’Regan, 2002). The mechanisms which determine the expression of the cholinergic phenotype have been investigated in primary cultures of sympathetic neurons of superior cervical ganglia (SCG) of newborn rats. These neurons are noradrenergic and maintain this phenotype in culture when grown alone. However, they can become cholinergic when cultured in the presence of nonneuronal cells or extracellular cholinergic differentiation factors such as leukemia inhibitory factor (LIF) or retinoic acid (RA) (Berrard et al., 1993; Patterson and Chun, 1974; Yamamori et al., 1989). Evidence was obtained that the expression of cholinergic properties in cultured SCG neurons is not mediated by a selective mechanism between two neuron populations, each producing a single neurotransmitter, but to a change in the identity of the neurotransmitter expressed in neurons that would otherwise have remained noradrenergic (Furshpan et al., 1986). Until now, the cholinergic differentiation of SCG neurons has mainly been associated with changes in synaptic vesicle cytochemistry and with the enhanced expression of ChAT and VAChT (Berrard et al., 1995; Johnson et al., 1980; Misawa et al., 1995), but to our knowledge, no information is available about the third protein required for cholinergic function, CHT1. In the present study, we thus investigated the regulation of HACU and CHT1-mRNAs when cultured SCG neurons acquire a cholinergic phenotype in response to LIF or RA. Surprisingly, our results indicate that high levels of HACU activity and CHT1mRNAs are expressed in SCG neurons grown in conditions where they develop mainly a noradrenergic phenotype. Moreover, HACU activity and CHT1-mRNAs are regulated in a manner similar to that of ChAT in response to LIF, but differently from that of ChAT in response to RA. In intact SCG, we found that CHT1-mRNAs are synthesized by the noradrenergic neurons. This suggests that CHT1 expression is not restricted to the cholinergic neurons in the peripheral nervous system. Results Cultured SCG neurons have high-affinity choline uptake activity We first assessed choline transport activity in SCG neurons grown without addition of any cholinergic differentiation factor (standard conditions) and which exhibit a predominantly noradrenergic phenotype. Kinetic analysis revealed that choline uptake is concentration-dependent and is saturated by increasing concentrations of choline, with an apparent Km value of c3 AM (Fig. 1A).

Moreover, choline uptake was strongly inhibited by the presence of HC-3 at concentrations that do not affect low-affinity choline transport (64% to 77% inhibition by 0.1 to 10 AM HC-3; Fig. 1B).

Fig. 1. Characterization of choline uptake system of neonatal SCG neurons grown in standard conditions. (A) Effect of choline concentration on choline uptake activity in neurons cultured for 7 days. Inset: Km value was determined using the Lineweaver-Burke transformation and was 3 AM in this particular experiment. (B) Hemicholinium (HC-3) sensitivity and Na+ and Cl dependence of choline uptake activity in neurons grown for 7 days. HC-3 was added at the indicated concentrations to the standard assay buffer containing 135 mM NaCl. Na+ and Cl dependency was investigated by replacing the NaCl by an equimolar concentration of either LiCl or NaI. Statistical analysis was performed using the unpaired Student t test: *P b 0.0008, with respect to uptake in the presence of NaCl. (C) Time course for choline uptake. Neurons were grown for up to 12 days, and choline uptake was measured at the times indicated either in the absence or in the presence of 10 AM HC-3. Total (x), HC-3-sensitive (E), and HC-3-resistant (n) uptake activities are represented. Data shown in (A–C) are the mean F SEM of triplicate determinations.

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The accumulation of [3H]choline was strongly reduced by replacing the NaCl in the transport assay buffer with equimolar amounts of either LiCl or NaI (95% or 65% inhibition, respectively; Fig. 1B). Results obtained with 10 AM HC-3 or in Na+-free conditions are in line with data reported by Bussie`re et al. (1995), but the levels of choline uptake inhibition were much higher in our experimental conditions. Choline transport activity in SCG neurons cultured in standard conditions is thus mediated in part by a high-affinity process, which displays all the major characteristics of the cholinergic transporter CHT1. A smaller proportion of choline is taken up by an additional process, which is unaffected by low concentrations of HC-3 or by a lack of Na+ or Cl and which thus fulfills some criteria of the low-affinity choline transport mechanism. In the following experiments, HACU refers to the 10 AM HC3-sensitive choline uptake activity. The time course for choline uptake in SCG neurons in culture is shown on Fig. 1C. Total uptake activity increased markedly with culture age (9.6- F 3.2-fold between days 3 and 12; mean of three independent experiments). The activity of the HC-3-insensitive form of choline uptake did not vary significantly during the course of culture, while HACU increased in a time-dependent manner similar to that of the total choline transport activity. During the first days of culture, HACU represented nearly half of the total transport activity measured (39.7% F 11.6% at day 4). This proportion increased with culture age, and after 1 week, HACU represented the major form of choline transport (63.7% F 9.2% at day 7 and 85.3% F 1.9% at days 12–14; mean of three experiments). This increase in HACU activity with age in cultures growing under standard conditions may be surprising. However, previous studies have demonstrated that dissociated SCG neurons grown in these conditions synthesize ACh, although at much lower levels than catecholamines (Patterson and Chun, 1977). We confirmed these data by measuring an increase of ChAT activity with time in sister cultures (data not shown). The development of cholinergic properties during the culture may be due to the influence of constituents of the culture medium: NGF, which permissively promotes cholinergic and catecholaminergic phenotypes, and a cholinergic phenotypeinducing activity present in adult rat serum (Chun and Patterson, 1977; Wolinsky and Patterson, 1985). However, 24 h after plating, no ChAT activity was detected, whereas HACU activity was clearly detectable and represented the same percentage of the total choline transport activity as at days 3–4 (data not shown). Differential regulation of high-affinity choline uptake activity by LIF and RA in cultured SCG neurons We next investigated the regulation of the HACU activity in cultured SCG neurons committed to the cholinergic phenotype by LIF or RA. A preliminary time course experiment revealed that significant HACU variations were measured after a 4-day treatment with LIF and after a treatment longer than 1 week for RA (data not shown). Thus, LIF or RA effects were analyzed in neurons treated for 4–5 or 9 days, respectively. In each experiment, ChAT activity, which is up-regulated by both factors, was measured as a positive control for the efficiency of the treatments. Addition of LIF on the third day of culture resulted in a dosedependent increase in HACU. The enhancement of HACU activity measured 4 days after LIF addition reached a plateau at a LIF concentration of about 10 ng/ml, with the half maximal saturation occurring at b3 ng/ml (Fig. 2A). As expected, ChAT activity also increased in response to LIF (Fig. 2A). HACU activity was 4.7-fold

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higher in cultures subjected to a 4-day treatment with 30 ng/ml LIF than in control sister cultures (Fig. 2B; result of six independent experiments). This LIF-induced enhancement was completely abolished in the absence of extracellular sodium (data not shown). In contrast, HC-3-insensitive uptake was 1.4-fold lower in LIFtreated cultures (Fig. 2B). This resulted in a 3.1-fold higher level of total choline uptake activity in response to LIF (Fig. 2B). In the presence of LIF, HACU represented 91% F 1% of total choline uptake activity, instead of 63% F 5% in control cultures (Fig. 2C). In contrast to LIF, RA was found to decrease HACU activity in a concentration-dependent manner, whereas, as shown previously, it induced simultaneously an increase of ChAT activity in the same culture (Fig. 3A). HACU activity was 1.7-fold lower in cultures treated for 9 days with 5 AM RA than in control cultures (mean of five independent experiments). HC-3-resistant uptake was less affected (1.2-fold lower than the control). In these conditions, total choline uptake was decreased by about 1.6-fold (Fig. 3B). In comparison to control cultures, and in contrast to the results obtained with LIF, RA did not significantly alter the level of HACU relative to total choline uptake (Fig. 3C). It should be noted that these results are discordant with those of Bussie`re et al. (1995), who reported that RA has no significant effect on choline uptake in cultured SCG. These discrepancies may be due to differences in experimental conditions (culture age, RA treatment, uptake assay). As RA treatment increases ACh content in cultured SCG neurons (Berrard et al., 1993; Bussie`re et al., 1995), our data suggest that choline supply is not the rate-limiting step for ACh synthesis in these neurons. Altogether, these results reveal that LIF and RA, both of which have been previously shown to induce the expression of ChAT and VAChT and raise ACh levels, differently regulate the activity of HACU in cultured sympathetic neurons. Expression of mRNAs encoding choline transporters in cultured SCG neurons To assess whether CHT1 mediates the HACU activity measured above, CHT1-mRNAs were analyzed by quantitative reverse transcription-polymerase chain reaction (RT–PCR) in untreated SCG neurons and when neurons become cholinergic under LIF or RA treatment. ChAT- and TH-mRNA levels, which are simultaneously increased and decreased by both factors, respectively (Cervini et al., 1994; Kobayashi et al., 1994), were also quantified as internal controls for up- and down-regulation triggered by these treatments. In SCG neurons grown for 8 days in the absence of LIF or RA, TH-mRNAs were 600-fold more abundant than ChAT-mRNAs (Fig. 4). This result is consistent with the predominantly noradrenergic phenotype of these neurons and validates our experimental conditions. However, like HACU, the levels of CHT1 transcripts were relatively high in these neurons. The amount of CHT1-mRNAs was 35-fold higher than that of ChATmRNAs and only 17-fold lower than that of TH-mRNAs. We then examined the effect of LIF and RA on CHT1-mRNA levels. When neurons were grown for 5 days in the presence of 30 ng/ml LIF, the expression of CHT1-mRNAs was up-regulated by 1.5-fold (Fig. 4A). In contrast, a 9-day treatment with 5 AM RA down-regulated by 2.8-fold the production of CHT1 transcripts (Fig. 4B). In comparison to control cultures, LIF- or RA-treated cultures had (i) 2.2- or 6-fold higher ChAT-mRNA levels, respectively, (ii) 3.8- or 1.4-fold lower TH-mRNA levels,

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Fig. 2. LIF up-regulates choline uptake activity in cultured SCG neurons. (A) Dose–response profiles of LIF-induced HACU and ChAT activities determined in sister cultures grown for 4 days in the presence of LIF at the indicated concentrations. Each data represent the mean F SEM of three determinations. (B) LIFinduced increase of choline uptake. Transport activities measured in LIF-treated cells are expressed relatively to those determined in untreated sister cultures (control). (C) Proportions of HACU and HC-3-resistant choline uptake in untreated neurons and neurons treated with LIF. In B and C, neurons were treated for 4 days by 30 ng/ml LIF. Each point represents the mean FSEM of the values obtained from six independent experiments, each performed in triplicate. Statistics were performed by comparing the mean choline uptake activities (B) or of the percentages of HACU and HC-3-resistant choline uptake activities (C) determined in LIF-treated and untreated cells, using the unpaired Student t test: *P b 0.02; #P = 0.0005 for both HACU and HC-3-resistant choline uptake.

respectively, and (iii) similar levels of mRNA encoding cyclophilin (Fig. 4). LIF and RA have thus opposing effects on CHT1 expression. The fact that CHT1-mRNAs are present in untreated neurons and regulated in a similar manner to HACU activity in response to LIF or RA suggests that the HACU activity measured in cultured SCG neurons can be attributed, at least in part, to the cholinergic transporter CHT1. The increase of CHT1-mRNA levels induced by LIF appears, however, to be lower than that of HACU activity reported above. This suggests that an additional transporter exists, which also takes up choline with high affinity, but which is more sensitive than CHT1 to LIF. Thus, we investigated whether CTL1 is also expressed in SCG neurons and regulated by LIF and RA (Fig. 4). RT–PCR analysis revealed the presence of CTL1-mRNAs in cultured SCG neurons. The CTL1-mRNAs are fourfold less abundant than CHT1-mRNAs in 1-week-old cultures. Moreover, both LIF and RA up-regulated CTL1-mRNA expression, the most efficient inducing factor being LIF [3.3-fold increase induced by LIF (Fig. 4A), instead of 1.9-fold obtained with RA (Fig. 4B)].

However, although CTL1 is able to transport choline with high affinity (O’Regan et al., 2000), it is unlikely that CTL1 is responsible for the majority of the HACU measured in our experimental conditions. First, CTL1-mediated choline uptake is only slightly affected by the absence of Na+ (O’Regan and Meunier, 2003), whereas 95% of the HACU is abolished in SCG neurons in Na+-free medium (Fig. 1B). Second, RA has opposite effects on the levels of HACU and CTL1-mRNAs. Thus, CHT1 may represent the main component of HACU activity determined in our conditions. Expression of mRNAs encoding CHT1 in intact SCG In the central nervous system, CHT1 and the corresponding mRNAs are specifically produced by the cholinergic neurons (Kobayashi et al., 2002; Kus et al., 2003; Lips et al., 2002; Misawa et al., 2001). Thus, the presence of high levels of CHT1-mRNAs in sympathetic SCG neurons grown in conditions in which they are predominantly noradrenergic was initially surprising. The synthesis

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Fig. 3. RA down-regulates HACU activity in cultured SCG neurons. (A) Dose–response curve of RA effects on HACU and ChAT activities determined on sister cultures. Neurons were grown for 9 days in the presence of the indicated concentrations of RA. Points represent the mean F SEM of three determinations. (B) Effect of RA on choline uptake. The transport activities are relative to those measured in sister cultures grown in the absence of RA (control). (C) Relative levels of HACU and HC-3-resistant choline uptake activities in untreated and RA-treated neurons. In (B) and (C), neurons were treated for 9 days by 5 AM RA. Data represent the mean F SEM of the results of five independent experiments, each performed in triplicate. Statistics were performed as noted on Fig. 2: *P b 0.03; ns: not significantly different (P = 0.4823 (B) or P = 0.5767 (C)).

of CHT1-mRNAs may have been induced by cultivating the cells. To test this possibility, the expression of CHT1-mRNAs was analyzed in intact SCG dissected from newborn or adult rats. Quantitative RT–PCR was first performed. CHT1-mRNAs were clearly detected in SCG, whereas ChAT-mRNAs were not. In both newborns and adults, CHT1-mRNAs were c40- and 60fold less abundant, respectively, than TH-mRNAs, which were produced at very high levels in these noradrenergic ganglia (Table 1). Moreover, in adults, the levels of CHT1-mRNAs were only fourfold lower in ganglia than in spinal cord (data not shown), a tissue rich in cholinergic cell bodies and in ChAT-mRNAs (Berrard et al., 1986). Thus, CHT1-mRNAs are expressed with relatively high abundance in SCG, which is consistent with the presence of HACU activity measured during the first stage of SCG neuron culture. Therefore, the presence of CHT1-mRNAs in cultured SCG neurons results from the normal in vivo transcription of the gene encoding CHT1 in these ganglia. We then investigated which types of cells express CHT1 in SCG. These ganglia are composed of noradrenergic neurons, small

intensely fluorescent (SIF) and nonneuronal cells. Morales et al. (1995) reported the identification of single ChAT-immunoreactive cell bodies in adult SCG; however, these ChAT-positive cells were not detected in other studies (Lindh et al., 1986). The possibility that only the nonneuronal cells of the ganglion synthesize CHT1mRNAs can be ruled out, as we have shown that these transcripts are expressed in cultured SCG neurons devoid of nonneuronal cells (Fig. 4). The same is true for SIF cells, which do not survive in dissociated SCG cell cultures in the absence of glucocorticoid (Doupe et al., 1985). It is also rather unlikely that the high levels of CHT1-mRNAs in SCG are produced by the reportedly small number of cholinergic cells that might be present in this ganglion. It is thus more probable that CHT1 is produced by the noradrenergic neurons of the ganglia. To test this possibility, the distribution pattern of CHT1-mRNAs was assessed by in situ hybridization. In situ hybridization revealed that CHT1-mRNAs are expressed by a large number of SCG cells (Fig. 5), which is consistent with the results of the quantitative RT–PCR analysis. The density of CHT1-positive cells was similar to that of TH-positive cells,

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M.-J. Lecomte et al. / Mol. Cell. Neurosci. 28 (2005) 303–313 Table 1 Qualification of CHT1-mRNAs, TH-mRNAs, and CTL1-mRNAs in intact SCG Newborn rats Adult rats

CHT1-mRNAs

TH-mRNAs

CTL1-mRNAs

6.2 F 0.8 3.1 F 0.5

258.8 F 23.5 191.6 F 11.1

14.1 F 2.9 11.5 F 1.6

Values are normalized to G3PDH mRNAs and given in arbitrary units.

confirming the specificity of the probes (data not shown). These results demonstrate that CHT1 gene transcription in the SCG occurs in noncholinergic cells. To further characterize the CHT1-synthesizing cells, we compared the expression of CHT1- and TH-mRNAs using a dual-labeling system. Most of the cells which express CHT1 (labeled in blue) were also TH-positive (labeled in red) (Fig. 5). The catecholaminergic cells of the SCG are either dopaminergic SIF cells or noradrenergic neurons. In neonatal rat, each ganglion contains approximately 200 SIF cells, which represents about 1% of the number of noradrenergic neurons (Davies, 1978; Era¨nko¨ and Soinila, 1981). As the proportion of double-labeled SCG cells observed in our study is much higher, our results demonstrate that the noradrenergic neurons synthesize CHT1 in SCG. To our knowledge, this is the first example of CHT1 expression in neuronal cells which are not cholinergic. In addition, in a small number of CHT1-positive cells, no THmRNAs was detected. Inversely, in some cells producing TH, no CHT1-mRNA was detected. These last results may be due to the synthesis of low levels of CHT1 (or TH) in cells producing high amounts of TH (or CHT1) or they may suggest that two subtypes of noradrenergic neurons exist in SCG: those producing CHT1 and those that do not. Further investigation is required to clarify this point. Taken together, these results reveal that the expression of CHT1 is not restricted to cholinergic neurons and suggest that CHT1 is not a cholinergic specific marker in the peripheral nervous system.

Discussion Fig. 4. Effect of LIF and RA on the levels of mRNAs encoding CHT1, ChAT, TH, and CTL1. Neurons were treated with 30 ng/ml LIF for 5 days (A) or with 5 AM RA for 9 days (B). Values represent either the abundance of each transcript normalized to that of G3PDH-mRNAs given in arbitrary units (histograms), or the variations in the levels of each transcript of interest in response to LIF (A) or RA (B) (tables). Note that, for space limitations, histograms representing results obtained for TH and cyclophilin have been divided by 10-fold. Data values are the mean F SEM of n quantifications, each performed in triplicate. Statistical analysis was carried out using the unpaired Student t test: *P b 0.03, ns: P = 0.2362. For the two by two comparison of CHT1-, ChAT-, TH-, and CTL1-mRNAs in cells grown in standard conditions, a value of P b 0.008 was always obtained.

although stronger signals were obtained with the TH probe. Note that the levels of expression vary considerably from cell to cell for both CHT1 and TH transcripts. In contrast, no ChAT-mRNAexpressing cell could be detected in our conditions (data not shown). The distribution patterns we obtained in the same conditions for the CHT1 and ChAT transcripts in rat brain were identical to those previously reported for cholinergic neurons,

Primary cultures of SCG neurons of newborn rats constitute a model system for investigating the mechanisms which determine the cholinergic phenotype. The emergence of cholinergic features in these neurons has been previously associated with the coordinated up-regulation of ChAT and VAChT expression (Berrard et al., 1995; Misawa et al., 1995). Here, we investigated the regulation of the third protein required for cholinergic presynaptic function, CHT1, when SCG neurons become cholinergic in the presence of LIF or RA. This study led to two major findings: (i) the cholinergic markers are differentially regulated by RA, which, in contrast to LIF, down-regulates HACU activity and CHT1-mRNA production, and (ii) CHT1 is present in the noradrenergic neurons of SCG, both in culture and in vivo. Until now, CHT1 has been exclusively identified in the cholinergic neurons of the central nervous system (Kobayashi et al., 2002; Kus et al., 2003; Lips et al., 2002; Misawa et al., 2001) and in some cholinergic nonneuronal cells (Haberberger et al., 2002; Fujii et al., 2003; Lips et al., 2003; Pfeil et al., 2003). Our study demonstrates for the first time that CHT1-mRNAs are expressed in neurons which produce a neurotransmitter other than ACh.

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Fig. 5. Detection of CHT1- and TH-mRNAs in newborn rat SCG. CHT1-mRNAs and TH-mRNAs were visualized by nonradioactive in situ hybridization. For the dual-labeling studies, CHT1 and TH transcripts were blue and red, respectively. No hybridization signal was observed in SCG when using the corresponding sense probes (data not shown). Scale bar = 50 Am.

The function of CHT1 in the noncholinergic neurons of SCG is still unclear. However, choline destined to phosphatidylcholine (PtdCh) synthesis has been shown to be imported not only by the low-affinity choline uptake system, but also by the high-affinity transport mechanism in SCG neurons (Bussie`re et al., 1995). Thus, one possible explanation for the presence of CHT1 in these neurons, which are devoid of ChAT, is an increased need for intracellular choline for membrane phospholipid synthesis. This possibility is conceivable in neonatal SCG and, in particular, in cultured neurons which have to regenerate their neurites damaged during cell dissociation. A correlation between the rate of choline uptake and that of neurite outgrowth in newborn rat SCG explants supports this assumption (Suidan and Tolkovsky, 1993). However, this hypothesis does not account for the expression of CHT1 in the neurons of adult ganglia. Moreover, we have shown that these neurons also express CTL1. This transporter takes up choline with high affinity and has been suggested to provide choline for new membrane synthesis in axotomized motoneurons (Che et al., 2002). CTL1 may thus be mediating activated choline transport for neurite membrane regeneration in SCG. Consequently, efficient choline uptake for membrane phospholipid synthesis in SCG neurons does not necessarily require the presence of CHT1. The transcription of the CHT1 gene in noradrenergic SCG neurons may also be explained by their phenotypic plasticity. These neurons, which can become cholinergic, may synthesize pools of CHT1 in order to respond rapidly to cholinergic environmental signals, since the presence of functional CHT1 at the plasma membrane would be delayed by its targeting, first to synaptic vesicles then to the plasma membrane (Ferguson et al., 2003; Ribeiro et al., 2003). Such prior synthesis is not required for ChAT, as this enzyme is active as soon as ChAT-mRNAs are translated. However, this hypothesis, which may account for the presence of CHT1-mRNAs in neonate SCG neurons at their terminal differentiation stage, does not explain the presence of these transcripts in adult mature noradrenergic neurons, in which such shifts in neurotransmitter type do not occur (Ross et al., 1977) or occur in vitro with considerably less efficiency than in neonatederived neurons (Potter et al., 1986). LIF and RA have been previously shown to trigger expression of the cholinergic phenotype in cultured SCG neurons, thus leading

to the enhancement of the ACh/norepinephrine ratio (Berrard et al., 1993, 1995; Cervini et al., 1994; Kobayashi et al., 1994; Misawa et al., 1995; Patterson and Chun, 1974; Yamamori et al., 1989). As choline uptake is considered as the rate-limiting step for ACh synthesis, the production of higher levels of ACh in response to LIF or RA treatment was expected to result from the increase of CHT1 expression and/or activity. The present study demonstrates that LIF up-regulates HACU activity and that the concentration dependence of this response was similar to that of the ChAT activity. In addition, LIF increases CHT1-mRNA levels. This confirms that LIF is a differentiation factor, which promotes the cholinergic phenotype by inducing the expression of each of the key proteins required for the cholinergic presynaptic function. These results also suggest the existence of common signaling pathways that regulate the expression of CHT1, ChAT, and VAChT under LIF treatment. However, LIF increases more strongly HACU activity than CHT1-mRNA levels. As CHT1 is mainly localized as a reserve pool in synaptic vesicles, one possible explanation for the lack of correlation between the variations in HACU activity and that of the CHT1-mRNAs is that LIF increases vesicular trafficking and thus CHT1 delivery to the plasma membrane. In contrast to LIF, RA down-regulates similarly both HACU activity and CHT1-mRNA levels, whereas it substantially upregulates ChAT activity and ChAT-mRNA levels in the same cells. Thus, RA differentially regulates the cholinergic markers. Distinct regulations of ChAT and HACU activities have been previously reported in other cellular models. For instance, the NGF-induced increase in HACU in the hippocampus of aged rats is delayed as compared to that of ChAT (Williams and Rylett, 1990). Other studies have described an up-regulation in HACU activity in situations where that of ChAT activity remained unchanged, for example, during the differentiation of cholinergic neuroblastoma cells or in the mouse brain after administration of a cyclic AMP analogue (Rylett et al., 1993; Vogelsberg et al., 1997). In these examples, differential regulation of HACU and ChAT was consistent with the notion that HACU is the ratelimiting step for ACh synthesis in the brain. In contrast, the decrease of HACU activity and CHT1 transcript levels and the simultaneous increase of ChAT activity and ACh synthesis levels

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question the view that choline uptake represents the ratecontrolling process for the ACh synthesis in cultured SCG neurons. This may be explained by the pool of CHT1 present in the noradrenergic neurons before they switch to the cholinergic phenotype. However, it should be noted that the presence of large amounts of CHT1 in neurons does not necessarily mean that each CHT1 molecule is functional and correctly targeted to the plasma membrane. LIF and RA have a differential effect on HACU activity and CHT1-mRNA levels, while both factors are also capable of inducing similar responses with respect to ChAT, VAChT (increase), and the catecholamine synthetic enzymes (decrease) in cultured SCG neurons. A previous study showed that both factors also have opposing effects on the norepinephrine transporter, whose expression was suppressed by LIF but stimulated by RA in cultured SCG neurons and PC12 cells (Matsuoka et al., 1997). Thus, RA appears to differentially regulate the expression of the neurotransmitter biosynthetic enzymes and the Na+-dependent transporters of the plasma membrane. It would be of interest to analyze whether this observation could be extended to other neurotransmitter systems. Several hypothesis could explain the effect of RA on choline uptake. Previous studies have shown that, in addition to extracellular sources of choline, endogenous free choline may be generated by hydrolysis of PtdCh via phospholipase D and used for ACh synthesis (Blusztajn et al., 1987; Lee et al., 1993; Yavin et al., 1989). This pathway may be activated under extreme conditions, such as dietary choline deficiency or when neuronal activity is high. As RA was found to activate phospholipase D in neuroblastoma cells (Antony et al., 2003), a first hypothesis would be that RA also increases PtdCh catabolism in SCG neurons, thus generating free intracellular choline. This postulated increase in PtdCh turnover would reduce the need for uptake of extracellular choline. Analysis of PtdCh levels in RA-treated cultures would allow to test this possibility. Alternatively, RA has been shown to down-regulate the expression of the neural a3subunit of the Na+-K+ ATPase in cell lines or in primary cultures of cardiac myocytes (Gilmore-Hebert et al., 1989; He et al., 1996). If such inhibition also occurs in SCG neurons, it would lead to a reduction in HACU activity, since choline uptake is powered by the Na+ electrochemical gradient generated across the plasma membrane by this Na+ pump.

Experimental methods

addition of 10 AM cytosine 1h-d-arabino furanoside (Sigma) for the first 5 days. All-trans RA (Sigma) or mouse recombinant LIF (R&D Systems) were added to the culture c60 h after plating. As RA was dissolved in ethanol, the corresponding control culture always contained an equal volume of ethanol, which never exceeded 0.1% of the volume of the culture medium. The culture medium was renewed every 2–3 days. For the ChAT assays and RNA preparations, cells were washed twice with PBS, harvested, collected by centrifugation, and stored at 808C until use. Choline uptake assay Choline uptake by SCG neurons was measured in culture wells essentially as described by Rylett et al. (1993). Cells were rinsed twice with Krebs-HEPES (KH) buffer (135 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 10 mM HEPES pH 7.4, and 10 mM glucose), then preincubated for 5 min in a water bath at 378C in 180 Al of either KH buffer or KH buffer with 17 AM HC-3 (ICN Biomedicals). Uptake was initiated by the addition of 120 Al of prewarmed KH buffer containing [3H]choline (79–84 Ci/mmol; final concentration 15 nM) after which incubation was continued for 10 min at 37 8C. We checked that choline uptake was linear over this period (data not shown). Saturation conditions were obtained by addition of unlabeled choline at the concentrations indicated (Fig. 1A). [3H]Choline uptake was stopped by the addition of 1 ml icecold KH buffer containing 1 mM HC-3. Culture dishes were then placed on ice. Cells were washed twice with ice-cold KH buffer, then disrupted in 300 Al 0.1 N NaOH. Aliquots (150 Al) of cell lysate were used for scintillation counting to determine the amount of [3H]choline incorporated. The protein content of the samples was quantified using 50 Al of the lysate. Background levels were determined by exposing parallel cultures to [3H]choline at 08C in the presence of 666 AM of nonradioactive choline. Choline uptake was normalized to sample protein content and expressed as fmol choline/Ag proteins/min, then values corresponding to the background were subtracted. Hemicholinium sensitive uptake, designated high-affinity uptake, was determined by calculating the difference between choline accumulation in the absence and in the presence of 10 AM HC-3. To measure the Na+- or Cl -independent choline uptake, the NaCl in the KH buffer was replaced with equimolar concentrations of LiCl or NaI, respectively. Each uptake measurement was performed in triplicate, and the values given are the mean F standard error of the mean (SEM).

Sympathetic neuron culture and treatments ChAT assay Primary cultures of SCG neurons from 2-day-old Wistar rats (Charles River) were prepared as previously described (Berrard et al., 1993; Hawrot and Patterson, 1979). For the determination of choline uptake and ChAT activity, dissociated cells (neuronal and nonneuronal) were plated onto 16-mm-diameter collagencoated plastic dishes (Falcon) at a density of about 6–8  104 cells, which corresponds to two ganglia, per well. Cells used for RNA preparation were cultured at the same density onto 35-mmdiameter dishes. Cultures were grown in 400 Al of bicarbonatebuffered Leibovitz’s L-15 medium (GIBCO Laboratories) supplemented with several additives including 50 ng/ml rat recombinant h-NGF (R&D Systems) and 5% heat-inactivated rat serum. Proliferation of nonneuronal ganglionic cells was prevented by

ChAT activity was determined as described by Fonnum (1975) using [3H]acetylcoenzyme A (4.9 Ci/mmol, Amersham) as a substrate. Activity was measured in homogenates obtained by vortexing cells in the presence of 0.2 M NaCl/0.2% (v/v) Triton X100. Each assay was done in triplicate. ChAT-specific activity was expressed as pmol/min/Ag proteins. Protein determination Protein content was measured according to Bradford (1976). Standard curves were generated using known concentrations of bovine serum albumin diluted in an equal volume of cell lysis

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solution (0.1 N NaOH or 0.2 M NaCl/0.2% (v/v) Triton X-100 for samples used for choline uptake or ChAT assays, respectively).

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the transcript of interest and for normalizing the results. All samples were amplified in duplicate or triplicate in each run. In situ hybridization

Reverse transcription and quantitative PCR Total RNA was purified by the RNable method (Eurobio) from SCG tissues and cultured neurons, or as described by Chirgwin et al. (1979) from rat spinal cord, and quantified spectrophotometrically. Single-stranded cDNAs were synthesized in a 20-Al reaction volume from 500 ng of RNA using 100 U of Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega) and random hexamers [pd(N)6, Amersham Pharmacia Biotech] as primers. Reactions without RNA or without reverse transcriptase were performed as controls. Real-time quantitative PCR was performed with a LightCycler thermal cycler system (Roche) using SYBR Green I. Reactions were carried out in glass capillaries in a total volume of 20 Al. The reaction mixtures contained 2 Al of the cDNA samples (diluted at least 10-fold), 3 mM MgCl2, 0.5 AM of each primer, and the LightCycler DNA Master SYBR Green I Mix (Roche), which provides Taq DNA polymerase, nucleotides, SYBR Green dye, and buffer. All PCRs were carried out using the following conditions: an initial 10-min denaturation step at 958C, followed by 40 cycles each consisting of a denaturation step at 958C (1 s), an annealing step at 628C (10 s), and an extension step at 728C (20 s). Fluorescence was measured at the end of each cycle. Negative controls (containing water or reverse transcription controls instead of cDNA) were included in each PCR run. After the 40th cycle, PCR specificity was checked by melting-curve analysis for all samples and controls. Relative quantifications were performed from the fluorescence measurements by comparison with a standard curve generated during the course of each run and given in arbitrary units. G3PDH was chosen as the standard because of its linear response over a large range of dilutions (10 1 to 10 5) of SCG cDNA preparations. Moreover, to correct for run-to-run and sample-to-sample variations in the amount of cDNA present or in PCR efficiency, mRNA levels were normalized to the levels of G3PDH mRNA determined from the same cDNA sample in the same PCR run. Its accuracy as the endogenous reference was checked by the absence of significant variations in the level of G3PDH mRNAs during LIF or RA treatment when cyclophilin was used as endogenous control (not shown). The following pairs of primers, chosen on different exons, were used for PCR: 5V-GAGGGCTCTACTCTGTGGCATA-3Vand 5V-TACTGTAGAACAATCGGGAGAA-3Vfor CHT1 (421 bp); 5VGGTGTGGTGTGTGAGCATTC-3V and 5V-GATGTTGTCCACCCGACCTT-3V for ChAT (383 bp); 5V-CGTAGCTGCACAGACATACCAT-3V and 5V-AATAGGTGCACTGGCTGGTACT-3V for CTL1 (420 bp); 5V-GGGCTATGTAAACAGAATGGGG-3V and 5V-AAAGGTTGGAGAAGGGGATGGA-3V for tyrosine hydroxylase (TH) (412 bp); 5V-AGGCTGTGGGCAAGGTCATC-3V and 5V-ATGGGGACTCCTCAGCAACT-3V for G3PDH (421 bp); and 5V-CCAGGATTCATGTGCCAGGG-3V and 5VGTGAGAGCAGAGATTACAGGG-3V for cyclophilin (409 bp). For each pair of primers, a series of cDNA template dilutions (10 1 to 10 4) was first analyzed to determine the one that gives optimal amplification. SCG cDNA preparations at the dilutions 10 2 or 10 1 were used to quantify TH or the other transcripts, respectively. The same cDNA dilution was used for amplifying

The intracardiac perfusion of newborn Wistar rats was performed using 0.1 M phosphate buffer pH 7.4 containing 0.9% NaCl (PBS) at room temperature, followed by 4% paraformaldehyde in PBS at 48C according to the guide lines of the French Animal Care Committee. SCG were dissected and postfixed in the same fixative overnight at 48C, then immersed in 15% sucrose/ PBS at 48C for 24 h. SCG were included in 7% gelatin/15% sucrose/PBS and cut serially on a cryostat into 14-Am-thick sections. Nonradioactive in situ hybridization was performed as described previously (Ravassard et al., 1997; Herzog et al., 2001). Riboprobes for CHT1 and TH were generated from PCR fragments subcloned into pGEMTeasy (Promega), whereas those for ChAT were generated from a cDNA (Brice et al., 1989). The CHT1 and TH fragments were amplified from cDNAs from rat spinal cord and adrenal medulla, respectively, with the following primer pairs: 5VAACGCAGCGAAGCCATCATAGT-3V and 5V-CCAATGCAAATGGCTGGTAGAG-3V for CHT1; 5V-CAGCTTGCACTATGCCCACC-3Vand 5V-CAGGGTAGTATAGAGCATGG-3Vfor TH. Plasmids were linearized and used as templates for the synthesis of sense or antisense riboprobes by T7 or SP6 RNA polymerases (Promega), respectively, in the presence of digoxygenin-UTP (Roche). For dual-labeled in situ hybridizations, either digoxygenin-UTP (for CHT1) or fluorescein-UTP (for TH) was used. Colorimetric revelations were performed with 5-bromo-4-chloro3-indolyl phosphate (Promega) and either nitroblue tetrazolium (Promega) for digoxygenin-UTP (blue), or 2-[4-iodophenyl]-3-[4nitrophenyl]-5-phenyl-tetrazolium chloride (Roche) for fluoresceinUTP (red).

Acknowledgments We are especially grateful to Seana O’Regan for many helpful discussions and critical reading of the manuscript, and Annie Lamouroux for assistance with PCR experiments. This work was supported by the Centre National de la Recherche Scientifique, the Universite´ Pierre et Marie Curie, the Fondation pour la Recherche Me´dicale, the Institut de Recherche sur la Moelle Epinie`re, the Association de Recherche sur le Cancer, and Aventis Pharma. M.J.L. was supported by the Association France Alzheimer.

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