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J Neurochem. Author manuscript; available in PMC May 20, 2009.
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Post-translational Regulation of an Aplysia Glutamate Transporter During Long-Term Facilitation

Abstract

Regulation of glutamate transporters accompanies plasticity of some glutamatergic synapses. The regulation of glutamate uptake at the Aplysia sensorimotor synapse during long-term facilitation (LTF) was investigated. Previously, increases in levels of ApGT1 (Aplysia glutamate transporter 1) in synaptic membranes were found to be related to long-term increases in glutamate uptake. In this study, we found that regulation of ApGT1 during LTF appears to occur post-translationally. Serotonin (5-HT) a transmitter that induces LTF did not increase synthesis of ApGT1. A pool of ApGT1 appears to exist in sensory neuron somata, which is transported to the terminals by axonal transport. Blocking the rough endoplasmic reticulum (RER)- Golgi-trans-Golgi network (TGN) pathway with Brefeldin A prevented the 5-HT-induced increase in ApGT1 terminals. Also, 5-HT produced changes in post-translational modifications of ApGT1 as well as changes in the levels of an ApGT1-coprecipitating protein. These results suggest that regulation of trafficking of ApGT1 from the vesicular trafficking system (RER-Golgi-TGN) in the sensory neuron somata to the terminals by post-translational modifications and protein interactions appears to be the mechanism underlying the increase in ApGT1, and thus, glutamate uptake during memory formation.

Keywords: Aplysia, glutamate transporter, trafficking, memory, post-translational modification

Introduction

Clearance of glutamate from the synaptic cleft occurs through diffusion and uptake of glutamate by high-affinity transporters. Consequently, glutamate transporters affect numerous aspects of synaptic transmission (reviewed in Danbolt, 2001; Huang and Bergles, 2004). Glutamate transporters are regulated by many factors at the transcriptional, translational and post-translational levels, but regulation of their trafficking to the membrane by phosphorylation and/or protein interaction also appears to be important (for review see Gegelashvili and Schousboe, 1997; Danbolt, 2001; Levenson, 2002b; Robinson, 2002, 2003; Gonzalez and Robinson, 2004a, 2004b).

Previously, we demonstrated that plasticity of glutamate uptake accompanies changes in synaptic efficacy and learning in Aplysia and the rat hippocampus (Levenson et al., 2000b; Levenson et al., 2002a; Pita-Almenar et al., 2006). In Aplysia, an increase in glutamate uptake in pleural-pedal synaptosomes parallels the occurrence of long-term facilitation (LTF) of the synapses between sensory neurons in pleural ganglia and their associated motor neurons in pedal ganglia, and long-term sensitization (LTS) of the tail-siphon withdrawal reflex (Levenson et al., 2000b). Regulation of glutamate uptake appears to be one of the expression mechanisms of LTS/LTF because both LTS/LTF and glutamate uptake are, to a large extent, co-regulated through the same signaling pathways (Khabour et al., 2004). Moreover, an increase in the high-affinity Aplysia glutamate transporter 1 (ApGT1) in pleural-pedal synaptic membranes appears to be responsible for the long-term increase in glutamate uptake associated with LTS (Collado et al., 2007). The long-term increase in ApGT1 protein in synaptosomes did not appear to be dependent upon an increase in ApGT1 gene expression or a translocation of ApGT1 from a synaptic vesicle pool to the plasma membrane (Collado et al., 2007).

In the present study, ApGT1 was located in sensory neuron cell bodies and varicosities and ApGT1 was synthesized in the cell body of sensory neurons and transported to the terminals by axonal transport. However, the increase in synaptosomal ApGT1 was not due to somatic or local newly synthesized ApGT1 but most likely due to transport of already synthesized ApGT1 to the terminals. Thus, we proposed that the transport of ApGT1 from a pre-existing pool in the somata of sensory neurons to the terminals was regulated by 5-HT. In support of this hypothesis, 5-HT produced a substantial decrease in the amount of labeled ApGT1 in the pleural ganglia. Moreover, blockade of the vesicular trafficking system [rough endoplasmic reticulum (RER)- Golgi-trans-Golgi network (TGN)] with Brefeldin A prevented the 5-HT-induced increase of ApGT1 in synaptosomes. Importantly, the pattern of post-translational modifications of ApGT1 was altered by 5-HT, indicating that phosphorylation and/or changes in glycosylation of ApGT1 occurred during LTF. Interestingly, 5-HT produced an increase in a newly synthesized protein named ACOP (ApGT1 co-precipitating protein), suggesting that ACOP may be involved in regulating ApGT1 during LTF. Our findings suggest that regulation of transport of proteins from the vesicular trafficking system to terminals by post-transcriptional modifications and/or protein-protein interactions is an important mechanism of regulation of membrane proteins involved in the expression of LTS/LTF.

Material and Methods

Pleural-pedal ganglia (8-10/group) were removed from Aplysia and incubated in isotonic L-15 (Atlanta Biologicals) and buffered filtered sea water (1:1) for 3 h prior to drug treatments in this media. After treatments, ganglia were maintained in culture media [L-15 and hemolymph (1:1)] at 15°C.

Cultures of Aplysia sensory neurons were prepared as previously described (Schacher and Proshansky, 1983; Chin et al., 1999; Levenson et al., 2000b) and incubated for 5 to 6 days in equal parts isotonic L-15 and hemolymph. Cultures with a minimum of 20 cells/plate were used in the experiments. Cultures were treated with drugs in a 1:1 mixture of isotonic L15 and BFSW. When isolated sensory neuron processes were used, the cell bodies of sensory neurons were removed by cutting the axon at a distance of about 50-70 μm from the cell body using a glass microelectrode and aspirating them with the microelectrode. Such isolated processes remain viable in culture for over 48 h (Schacher and Wu, 2002; Liu et al., 2003).

To obtain large membranes, tissues were homogenized in grinding buffer (GB; 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM Tris, pH 7.5 and protease inhibitor cocktail) and centrifuged (16,000 g, 5 min) at 4 °C to pellet large membranes. The resulting pellet was resuspended in 1% SDS with 10 mM Tris (pH 7.5) or lysis buffer (see below) to give “large membranes”. Synaptosomes were immediately prepared after removal of ganglia as previously described (Chin et al., 1989; Chin et al., 1990; Levenson et al., 2000b).

Prior to 35S incorporation, tissues were incubated in “cold” media (L-15 without methionine and cysteine, Atlanta Biologicals) and BFSW (1:1) for 3 h. To label newly synthesized proteins, tissues were then incubated for 12 h in 3-5 ml of culture media with 35S methionine and 35S cysteine (0.5 mCi/ml; Perkin Elmer) at 15 °C. During chase periods, ganglia were incubated in “cold” media. After labeling and chase periods, synaptosomes or membrane proteins were prepared and ApGT1 was purified by Immunoprecipitation (IP).

To obtain ApGT1, membrane protein fractions were lysed in an IP lysis buffer (1 mM Na3VO4, 10 mM Na2HPO4, 1% Triton X-100, 20 mM NaF, 50 mM NaCl, 20 mM Tris plus protease inhibitor cocktail, pH 7.6). Cell lysate protein (15 - 25 μg) was preadsorbed for 1 h with Protein G beads (Sigma) and subsequently incubated at 4°C overnight with anti-ApGT1 antibody. Antigen-antibody complex was bound by Protein G- sepharose at 4°C for 3h. Precipitated proteins were dissociated by incubation with sample loading buffer (3% glycerol, 9% SDS, 15% 2-mercaptoethanol, 0.2% bromophenol blue, 375 mM Tris, pH = 6.8) and boiling for 10 min. Protein samples were resolved via SDS-PAGE, transferred, and blotted using the ApGT1 antibody. After Western blotting, the membranes were exposed to X-ray film to detect only 35S labeled protein. Blotting of the membrane did not interfere with subsequent autoradiography.

Two-dimensional gel electrophoresis was performed (O’Farrell, 1975) by Kendrick Labs, Inc. (Madison, WI). Isoelectric focusing was carried out using 1% pH 3.5-10 and 1% pH 2.5-5 ampholines or 2% pH 3.5-10 ampholines (GE Healthcare, Piscataway, NJ) for 20,000 volt-h. SDS slab gel electrophoresis (10% acrylamide) was carried out for about 5 h at 25 mA/gel. The gel was then transferred onto PVDF membranes overnight at 225 mA and approximately 100 volts/ two gels and blotted for ApGT1.

Glutamate Uptake, Western blots, immunostaining procedures and statistical analysis are described in the Supporting information.

Results

5-HT treatments of isolated pleural-pedal ganglia increased ApGT1 in synaptosomes

To develop an in vitro system to study regulation of ApGT1, we tested whether ApGT1 was increased in synaptosomes from pedal ganglia 24 h after exposure of isolated pleural-pedal ganglia to 5-HT (2 h, 50 μM), a treatment that mimics the effects of behavioral training and leads to the induction of LTF (Emptage and Carew, 1993; Zhang et al., 1997). 5-HT increased the levels of ApGT1 in pedal synaptosomes 24 h after treatment (61 ± 23%, n = 11, one-tailed t-test, t = 2.706, p = 0.01; Fig. S1). The result of this experiment further suggests that an increase in ApGT1 is responsible for the increase in glutamate uptake (Levenson et al., 2000b; Khabour et al., 2004).

ApGT1 localizes in pleural sensory neuron cell bodies and varicosities

To study regulation of ApGT1, we determined the locus of ApGT1 expression and synthesis, its half-life and how ApGT1 was transported to synaptic terminals. ApGT1 was localized in pleural-pedal ganglia by immunofluorescence using an anti-ApGT1 antibody (Collado et al., 2007). DAPI staining identified nuclei. High levels of ApGT1 immunoreactivity were found surrounding the nucleus of small sensory neurons in the pleural ganglia (SNs, see cells inside circle, Fig. 1a and cells labeled as SN, Fig. 1c) and in fibers coming from the sensory neuron cluster (arrowheads, Fig. 1a and c). ApGT1 staining in sensory neuron cell bodies appeared to be in the plasma membrane and in association with internal membranes, presumably RER and/or Golgi, but not in the nucleus. In addition, high levels of ApGT1 immunoreactivity was associated with glial cells (small nuclei, see white arrows) surrounding neurons as well as with processes (arrowheads) and/or glia (arrows) in connectives and neuropil (Fig. 1a and c). This pattern of staining correlates with the in situ hybridization pattern of ApGT1 mRNA (Levenson et al., 2000a). The immunostaining for ApGT1 was specific because cells from the sheath surrounding the ganglion showed no staining (see yellow arrows, Fig. 1a and b) as expected from in situ hybridization studies (Levenson et al., 2000a). Moreover, tissue stained under the same conditions but lacking primary antibody did not show staining (Fig. 1e). Thus, in pleural ganglia, ApGT1 appears to be found in sensory neuron cell bodies and processes as well as in glial cells.

Fig. 1
Localization of ApGT1 in pleural-pedal ganglia

In pedal ganglia, some ApGT1 staining was found surrounding pedal neurons (PN, Fig. 1b), but no staining was observed surrounding the nucleus of these large cells suggesting that ApGT1 was not in the soma of these neurons (see N1, Fig. 1d). The examination of stained pedal ganglia at high magnification (60x) revealed that ApGT1 was found associated with small nuclei (glial cells, see white arrows) surrounding the large neuronal cell bodies and with glial indentations into the neuronal cell bodies (see red arrows pointing to glial indentations into cell N2), in agreement with previous morphological studies (Coggeshall, 1967). High intensity staining for ApGT1 was also found associated with glial cells (white arrows) in connectives and the periphery of the neuropil (data not shown). Staining inside the neuropil of pedal ganglia was associated with glia but some fiber-like staining was also observed (bottom left corner Fig. 1b, data not shown). Thus, in pedal ganglia, immunoreactivity for ApGT1 was mostly associated with glial cells and presumably processes of sensory and/or other neurons in the neuropil.

To compare levels of ApGT1 between different compartments in pleural-pedal ganglia, levels of ApGT1 were measured by western blot in sensory neuron clusters (where sensory neuron cell bodies are located), pedal neuropil (where processes of sensory neuron are located) and the remainder of pleural or pedal tissue. Levels of ApGT1 were much higher in tissues from pleural ganglia than in tissues from pedal ganglia (Fig. 1f). Moreover, ApGT1 was much more concentrated in sensory neuron clusters than in the pedal or pedal neuropil, demonstrating that there are high levels of ApGT1 in sensory neuron cell bodies consistent with a large pool of ApGT1 available in somas but not processes of sensory neurons.

The subcellular distribution of ApGT1 was studied in isolated cultured sensory cells. Results from 30 cultures (a total of 80 pleural sensory cells) showed that ApGT1 was in the sensory neuron cell body as well as in varicosities (Fig. 2a, arrowheads). ApGT1 immunoreactivity was not detected in the nucleus of sensory neurons (SN, Fig. 2b1). Immunostaining for ApGT1 was brighter in the sensory neuron cell body than in the processes or varicosities (arrowheads), suggesting again that a pool of ApGT1 exists in the sensory neuron cell body available for transport. Cultures that were not incubated with primary antibody and did not show any staining (n = 7 cultures). Moreover, the immunostaining for ApGT1 was competed out by preadsorption with the antigen peptide (n = 5 cultures, Fig. 2c). These results from cultured sensory neurons support our data on pleural-pedal ganglia suggesting that ApGT1 is found in cell bodies and terminals of pleural sensory neurons.

Fig. 2
ApGT1 immunostaining of cultured sensory neurons

ApGT1 was synthesized by pleural sensory neurons and transported to terminals in pedal ganglia

To determine the locus of ApGT1 synthesis, isolated pleural ganglia and isolated pedal ganglia were incubated with radioactive methionine and cysteine for 12 h. Since pleural ganglia yield very little protein, the entire sample was used to start immunoprecipitations (IPs). The amount of newly synthesized ApGT1 relative to total ApGT1 was 8-fold greater in pleural ganglia than in pedal ganglia (Fig. 3a). Also, the amount of newly synthesized ApGT1 relative to total ApGT1 was 4-fold greater in isolated pleural sensory neuron clusters (SN clusters) than in the remaining pleural ganglia tissue after removal of sensory clusters (Re pleural) (Fig. 3b). These results suggest that the rate of synthesis of ApGT1 is lower in glia than in sensory neurons. Thus, newly synthesized ApGT1 observed in sensory clusters was most likely due to ApGT1 synthesized by pleural sensory neurons.

Fig. 3
ApGT1 is synthesized by sensory neurons

What is the source of ApGT1 in synaptosomes (terminals) from pedal ganglia? We hypothesized that ApGT1 was synthesized in somata of sensory neurons in the pleural ganglia and transported to terminals in the pedal ganglia. To test this hypothesis, labeled ApGT1 was measured in pedal synaptosomes (a preparation enriched in neuronal terminals and almost devoid of glial material, Levenson et al., 2000b) using a pulse-chase protocol. Isolated pleural-pedal ganglia were incubated with 35S-methionine and cysteine for 12 h, at which point the protein synthesis inhibitor anisomycin (20 μM) was added to the bath. Some ganglia were lysed immediately after the 12 h incubation in label (0 h), and the rest after a 6 or 12 h chase period in the presence of anisomycin (Fig. 4a). Levels of newly synthesized ApGT1 were normalized to levels of total ApGT1 in synaptosomes, which remained unchanged in all groups compared to the 0 h time point (two-tailed t-test, all groups p > 0.05). Newly synthesized ApGT1 was detected in synaptosomes after 12 h of incubation (Fig. 4a). The newly synthesized ApGT1 might be due to ApGT1 synthesized in the soma and transported to the terminals or synthesized locally at the terminals. The lag period of 12 h in theory allows ApGT1 to be synthesized in the pleural ganglia and transported to the terminals of the pedal ganglia (Goldberg et al., 1981). Labeled ApGT1 in synaptosomes increased after chase periods of 6 and 12 h in the presence of anisomycin. The level of labeled ApGT1 was significantly increased by 3.5-fold after the 12 h chase period (One sample t-test, t = 3.216, df = 7, p < 0.02; Fig. 4a and b). Because protein synthesis was inhibited during the chase period, the increase in ApGT1 was most likely due to transport of previously synthesized ApGT1 to the terminals. This result also indicates that ApGT1 is not being degraded; therefore, the half-life of ApGT1 at the terminals may be longer than 12 h. A long half-life of ApGT1 is also supported by experiments showing no decrease in glutamate uptake after treatment of pleural-pedal ganglia with anisomycin for 24 h and 72 h (Khabour and Eskin, unpublished observations).

Fig. 4
ApGT1 is transported from pleural ganglia to pedal synaptosomes by fast axoplasmic transport

In Aplysia, the anterograde transport of vesicles containing glycoproteins occurs by fast axonal transport through microtubules at a rate of approximately 100-170 mm/day (Goldberg et al., 1981). Does the increase in ApGT1 in synaptosomes after the chase period result from microtubule-dependent transport? To examine this issue, colchicine (10 mM), an inhibitor of microtubule polymerization in Aplysia (Goldman et al., 1976), was added after 12 h of incubation in label and labeled ApGT1 was measured in pedal synaptosomes 12 h later. The addition of colchicine completely blocked the increase in labeled ApGT1 seen after a 12 h chase period (One-tailed t-test with Welch’s correction, t = 2.785, df = 8, p < 0.01; Fig. 4a and c). This result indicates that the increase in ApGT1 in pedal synaptosomes after the chase period is due to microtubule-dependent axonal transport.

To further test whether ApGT1 synthesized in the pleural ganglia is the source of the ApGT1 transported to pedal terminals, pleural-pedal ganglia were incubated in label for 12 h, and then pleural ganglia were surgically removed from pedal ganglia and isolated pedal ganglia were further incubated in label for 12 h in the presence of anisomycin. Removing pleural ganglia before the chase period significantly reduced the increase of ApGT1 in pedal synaptosomes observed 12 h after the chase period in attached pleural-pedal ganglia (Fig. 4c; One-tailed t-test with Welch’s correction, t = 2.046, df = 8, p < 0.04). A small amount of transport to terminals appeared to occur during the chase period even after pleural ganglia were removed (Fig. 4c). However, the magnitude of the increase of labeled ApGT1 in isolated pedal ganglia was reduced by 170 % when pleural ganglia were removed from pedal ganglia during the chase period. Some percentage of the increase seen within the pedal ganglia after removal of the pleural ganglia was likely due to labeled ApGT1 already in the pleural-pedal connectives that then continued transport to the terminals during the chase period. In summary, ApGT1 appears to be synthesized in the cell body of pleural sensory neurons and transported to the terminals in pedal ganglia by axonal transport. Also, ApGT1 appears to have a long half-life in the terminals.

The increase of ApGT1 in synaptic terminals after 5-HT was not due to an increase in translation of ApGT1

Inhibition of protein synthesis during induction of the long-term increase in glutamate uptake blocked the increase in glutamate uptake (Levenson et al., 2000b; Collado et al., 2007). However, the requirement for protein synthesis for the increase in glutamate uptake does not necessarily reflect an increase in the translation of the transporter, i.e., expression, but rather may be part of the induction mechanisms underlying the increase in glutamate uptake. We investigated whether 5-HT increases ApGT1 translation by measuring labeled ApGT1 in pedal synaptosomes after 5-HT treatment of isolated pleural-pedal ganglia. This experiment was designed (Fig. 5a) based on our previous results indicating that protein synthesis is not required for the 24 h increase in glutamate uptake by 7.5 h after the beginning of 5-HT treatment (Collado et al., 2007) and that at least 12 h are needed for newly synthesized protein to accumulate in the terminals (Fig. 4). In this experiment, immunoprecipitation procedures were begun with the same amount of protein in all groups (Fig. 5b).

Fig. 5
Synthesis of ApGT1 protein was not regulated by 5-HT

If the increase in ApGT1 was due to an increase in the synthesis of ApGT1 either in the soma or terminals, then 5-HT treatments should increase the amount of label incorporation into ApGT1 in pedal synaptosomes. No significant change in labeled, newly synthesized ApGT1 was observed in pedal synaptosomes 20 h after 5-HT treatment (-16 ± 15 %, n = 4; one-tailed t-test, t = 1.072, p = 0.18; Fig. 5b and c). However, in these same samples, total ApGT1 was significantly increased in pedal synaptosomes both before IP (65 ± 29 %, n = 4, one-tailed t-test, t = 2.270, p < 0.05) and after IP (53 ± 18 %, n = 6, one-tailed t-test, t = 4.541, p < 0.004; Fig. 5b and c). Thus, the 5-HT-induced increase in ApGT1 in synaptosomes does not appear to be due to an increase in synthesis of ApGT1 in terminals or somata but rather to more ApGT1 transported from a pre-existing pool in the soma of sensory neurons to their terminals. Alternatively, the rate of endocytosis or degradation of ApGT1 could be down regulated by 5-HT. However, since the half-life of ApGT1 appears to be long (Fig. 4 and Khabour and Eskin, unpublished observations), it is more likely that trafficking of ApGT1 rather than turnover of ApGT1 in the terminals is regulated during induction of LTF.

It is possible that a change in synthesis of ApGT1 could be detected in the pleural ganglia but not in pedal ganglia. Therefore, levels of labeled, newly synthesized ApGT1 were measured in total membranes of pleural ganglia using the same experimental design (Fig. 5a). A significant decrease in labeled, newly synthesized ApGT1 was observed in pleural ganglia treated with 5-HT compared with non-treated ganglia (-46 ± 9 %, n = 4, two tailed t-test, p < 0.02, Fig. 5d). This result suggests that synthesis of ApGT1 was not increased by 5-HT but the pool of labeled ApGT1 in the pleural ganglia appears to be reduced. The decrease in labeled ApGT1 in the pleural was observed 20 h after 5-HT treatment, so it is possible that the newly synthesized labeled ApGT1 was transported to the terminals subsequent to the transport of ApGT1 from an unlabeled pool. The transport of newly synthesized ApGT1 probably occurred later than unlabeled ApGT1, which may already be in the TGN or transport vesicles. This would explain why labeled ApGT1 did not increase in the pedal synaptosomes by 20 h. These results strongly suggest that the pool from which ApGT1 is transported resides in the soma of sensory neurons.

Interestingly, a 35S-labeled 60-65 KDa protein that runs just below ApGT1 and was not recognized by the ApGT1 antibody was observed in autoradiographs from IPs in all the fractions studied (membranes from pleural and pedal ganglia, sensory neuron clusters and synaptosomes, e.g., see Figs. Figs.3a3a and and4a).4a). This protein that consistently, and specifically, co-immunoprecipitated with ApGT1 was named ACOP (ApGT1 co-precipitating protein). 5-HT significantly increased the level of 35S-labeled co-immunoprecipitated ACOP in synaptosomes 20 h after treatment (37 ± 11 %, n = 6, two-tailed t-test, t = 3.370, p < 0.02; Fig. S2). These results suggest that ACOP might be involved in the trafficking or regulation of ApGT1. Although identification of ACOP was attempted, ACOP’s identity remains unknown.

The pleural ganglia as well as sensory neuron cell bodies are required for the transcription-dependent long-term increase in glutamate uptake

Previous experiments suggested that a pool of ApGT1 in sensory neuron cell bodies might be the source of the increase in ApGT1 by 5-HT. To determine whether sensory neuron cell bodies were required for the increase in glutamate uptake, isolated pedal ganglia were treated with 5-HT. Treatment of isolated pedal ganglia with 5-HT (50 μM, 1.5 h) led to an increase in glutamate uptake 24 h later (27 ± 7%, n = 13, p < 0.05, Fig. 6a). The amplitude of the increase in glutamate uptake was lower than that produced in the presence of sensory neuron cell bodies (45 ± 8% versus 27 ± 7% respectively, Fig. 6a). Moreover, treatment of isolated pedal ganglia with the transcription inhibitor DRB (100 μM, added 30 min prior to 5-HT treatment and throughout the experiment) did not affect the increase in glutamate uptake produced by 5-HT on isolated pedal ganglia. (31 ± 8%, n = 9, p > 0.05, Fig. 6a) The basal levels of glutamate uptake (3 ± 3%, n = 6, p > 0.05, Fig. 6a) were also not affected by DRB. In contrast, the increase in glutamate uptake produced in vivo by electrical stimulation and in vitro by exposing isolated pedal-pleural ganglia or cultured sensory neurons to 5-HT was completely inhibited by DRB (Levenson et al., 2000b; Khabour and Eskin, unpublished). These results suggest that cell bodies of sensory neurons are required for obtaining the transcription-dependent full increase in glutamate uptake measured in pedal synaptosomes.

Fig. 6
Somata of sensory neurons are required for the transcription dependent increase in glutamate uptake by 5-HT

We also investigated the role of sensory neuron cell bodies in eliciting changes in glutamate uptake in sensory neurons in culture. Sensory neuron cell bodies were removed and processes were then exposed to 5-HT (50 μM, 1.5 h) 2-3 h later. Aplysia neuronal processes in culture have been shown to remain healthy and functional at least 48 h after the removal of cell bodies (Schacher and Wu, 2002; Liu et al., 2003). Removal of the sensory neuron cell bodies from cultured sensory neurons decreased glutamate uptake by approximately 50% because glutamate transporters are also localized in the plasma membrane of cell bodies (data not shown). 5-HT did not induce significant changes in glutamate uptake in neuronal processes measured 24 h after the 5-HT treatment (13 ± 11%, n = 10, p > 0.05, Fig. 6b). In contrast, treatment of intact cultured sensory neurons with 5-HT produced a long-term increase in glutamate uptake measured 24 h later (72 ± 14%, n = 8, p < 0.01, Fig. 6b). Thus, the presence of sensory neuron cell bodies is required for the production of the long-term increase in glutamate uptake in cultured sensory neurons. This result also indicates that the pool of transporters required for the change in glutamate uptake resides in intracellular stores in the soma of sensory neurons.

The long-term increase of ApGT1 in pedal synaptosomes appeared to require transport through the RER-Golgi-TGN network

Lack of transcriptional or translational regulation together with other results led us to hypothesize that regulation of the transport of ApGT1 from the vesicular trafficking system (RER-Golgi-TGN) to terminals by 5-HT is responsible for the long-term increase of ApGT1 in synaptosomes. To test this, isolated pleural-pedal ganglia were exposed to 5-HT (1.5 h, 50 μM), and then to Brefeldin A (18 μM) for 22.5 h. Brefeldin A, an inhibitor of COPI and some clathrin-mediated endocytosis, blocked the long-term increase in glutamate uptake (Levenson et al., 2000b). Brefeldin A applied alone for 22.5 h did not affect the levels of ApGT1 in pedal synaptosomes (7.1 ± 6.8 %; n = 7; one sample t-test, t = 1.041, p = 0.34; Fig. 7), further supporting that ApGT1 has a long half-life at the terminals. As previously shown, levels of ApGT1 were increased in pedal synaptosomes when isolated pleural-pedal ganglia were treated with 5-HT (39.4 ± 2.4 %; n = 4; one sample t-test, t = 16.18, p = 0.0005; Fig. 7). Brefeldin A added to pleural-pedal ganglia after 5-HT treatment blocked the long-term increase in ApGT1 (14.3 ± 5.2 %; n = 5; One-way ANOVA followed by Tukey’s post hoc test: F = 6.887, df = 15, p = 0.009, Brefeldin A vs 5-HT: p < 0.01, Brefeldin A vs 5-HT + Brefeldin A: p > 0.05, 5-HT vs 5-HT+ Brefeldin A: p < 0.05; Fig. 7). Therefore, transport of ApGT1 through RER-Golgi-TGN appears to be required for the long-term increase in ApGT1 in synaptic terminals. Brefeldin also disrupts the cycling of proteins through the endosomal system but it seems unlikely that ApGT1 is trafficked through this system.

Fig. 7
RER-Golgi-TGN transport is required for the long-term increase in ApGT1 by 5-HT

Most ApGT1 in the pleural ganglia appears to be mature since ApGT1 was EndoH insensitive while PGNase F reduced the molecular weight of ApGT1 from ~ 70 KDa to ~60 KDa in the pleural demonstrating that ApGT1 is indeed a glycoprotein (Collado, MS and Eskin, A, unpublished results). This difference in molecular weight is consistent with the five N-linked oligosaccharide sites in ApGT1 containing complex oligosaccharides. This result suggests that the ApGT1 pool in sensory neuron cell bodies might be located past the RER, probably in the trans-Golgi and/or transport vesicles as observed for other amino acid transporters (Hatanaka et al., 2006).

5-HT produced post-translational changes of ApGT1

Several consensus glycosylation and phosphorylation sites are present in the predicted protein sequence of ApGT1 (Collado et al., 2007). Therefore, if exit of ApGT1 from the RER-Golgi-TGN system is regulated by 5-HT, then a change in the phosphorylation pattern and/or a change in ApGT1 maturation (indicated by differential glycosylation) should be observed (Lodish et al., 2008). To test whether ApGT1 was regulated post-translationally by 5-HT, shifting of ApGT1 on high-resolution two-dimensional polyacrylamide gels (2D gels) blotted with ApGT1 was studied.

ApGT1 exhibited extreme charge heterogeneity, a pattern very characteristic of glycoproteins that can result from phosphorylation, glycosylation and/or other post-translational modifications (Dunbar, 1987). The predicted molecular weight of ApGT1 based upon its sequence is 62 kDa. However, ApGT1 exhibited a moderate molecular weight heterogeneity ranging approximately from 70 kDa to 75 kDa (Fig. 8a) that could result mainly from differential glycosylation and to a less extent, other types of post-translational modifications like phosphorylation which can account only for around 100 Daltons per phospho-site.

Fig. 8
5-HT induced changes in post-translational modifications of ApGT1

Isolated pleural-pedal ganglia were treated with 5-HT for 2 h and prepared for gel analysis 24 h later. Some charge isoforms of ApGT1 disappeared or decreased after 5-HT treatment (spots # 8, 9, 10 and 11; Fig. 8a and b) and some new charge isoforms of ApGT1 appeared or increased after 5-HT treatment (spots # 1, 2, 3, 4, 5, 6, 7; Fig. 8a and b). This experiment was repeated 3 times with similar results. The changes observed were mainly due to changes in charge while changes in molecular weight were not apparent. Overall, it appeared that most charge isoforms of ApGT1 were shifted from basic to acidic isoforms by 5-HT (Fig. 8b). The approximately 0.1 pH differences between protein spots observed in the 2D gels are indicative of phosphorylation isomers. Moreover, about 9 protein spots are visible on the 5-HT 2D gel, consistent with 9 consensus phosphorylation sites found in ApGT1. This post-translational regulation produced by 5-HT suggests that transport of ApGT1 to the terminals may be regulated by phosphorylation.

Discussion

We have extended the knowledge of the regulation of ApGT1 during LTF and found that the increase in ApGT1 in synaptosomes is induced and expressed in isolated pleural-pedal ganglia treated with 5-HT. Therefore, ApGT1 and glutamate uptake increase in synaptosomes, but not in the cell/glial fraction, from isolated ganglia treated with 5-HT or intact animals exposed to either electrical stimulation or 5-HT (Levenson et al., 2000b and Collado et al., 2007). Therefore, the regulation of levels of ApGT1 at the synaptic membrane appears to be the mechanism of expression of the long-term increase in glutamate uptake.

Although the long-term increase in glutamate uptake is dependent on macromolecular synthesis, neither transcription nor translation of ApGT1 is regulated during LTS/LTF (Collado et al., 2007 and Fig. 5). How can the requirement of transcription and translation for the increase in glutamate uptake (Levenson et al., 2000b) be explained in light of a lack of a change in synthesis of ApGT1? One possibility is that the requirement for transcription and translation is for the induction phase of the increase in glutamate uptake. Another possibility is that the increase in glutamate uptake could be dependent on the transcription of genes or translation of proteins other than ApGT1, which may be important for the expression of the change in glutamate uptake. An example of such proteins could be glutamate transporter interacting proteins (e.g., targeting proteins), which might regulate ApGT1 transport to the terminals (for review see Gonzalez and Robinson, 2004b). 5-HT induced an increase in newly synthesized levels of ACOP, a protein that co-immunoprecipitates with ApGT1 (Fig. S2). Thus, an increase in the translation of ACOP could be one source of the requirement for translation.

What is the cellular source of increased ApGT1 in synaptic membranes? In this study, we showed that high levels of ApGT1 were found in sensory neuron cell bodies in the sensory cluster (Fig. 1) as well as in cell bodies and varicosities of cultured sensory neurons (Fig. 2). Because the somata of sensory neurons synthesize ApGT1 and the somata contain ApGT1, pleural sensory neurons appear to be a source of glutamate transporters (Fig. (Fig.11--33 and Collado et al., 2007). Moreover, ApGT1 is transported from pleural ganglia to pedal terminals in a microtubule-dependent manner (Fig. 4). Although high levels of ApGT1 are also found in glial cells in pleural-pedal ganglia, glia do not appear to be the source of the additional ApGT1 since the change in levels of ApGT1 and glutamate uptake is specific to the synaptosomal fraction (which is enriched in neuronal terminals and almost depleted of glia) and not observed in the cell/glial fraction (Levenson et al., 2000b; Collado et al., 2007). Importantly, increases in glutamate uptake produced by 5-HT occur in cultures of isolated sensory neurons that contain few if any glial cells. Furthermore, glia do not appear to synthesize ApGT1 at high rates (Fig. 3). Labeled ApGT1 measured in our pulse-chase experiments most likely corresponded mainly to ApGT1 synthesized in sensory neurons.

The 5-HT-induced increase in ApGT1 may be due to trafficking of ApGT1 from a reserve pool in somata of sensory neurons to the terminals. First, the higher rate of synthesis and higher levels of ApGT1 found in the somata of sensory neurons than in pedal ganglia, neuropil, and processes of cultured sensory neurons (Figs. (Figs.11--3;3; Collado et al., 2007) suggest that a reserve pool of ApGT1 exists in the somata rather than in the processes of sensory neurons. Second, pleural ganglia (presumably sensory neuron cell bodies) appear to be the main source of ApGT1 transported to the terminals (Fig. 4c). Third, the increase in the levels of ApGT1 observed in synaptosomes of pedal ganglia was not due to translation of ApGT1 (Fig. 5) or to translocation from a synaptic vesicle pool to the membrane (Collado et al., 2007). Also, 5-HT produced a substantial decrease in the amount of labeled ApGT1 in the pleural ganglia appearing to reduce the pool in the pleural (Fig. 5d). Finally, pleural ganglia and cell bodies of isolated sensory neurons are required for the full, normally transcription-dependent increase in glutamate uptake by 5-HT (Fig. 6). What is the source of the non-transcription dependent increase in uptake observed in isolated pedal ganglia (Fig. 6a)? These glutamate transporters may come from a pool existing in the pedal ganglion that can be activated upon removal of the pleural ganglia.

The reserve pool of ApGT1 may be located in the Golgi network in the somata of sensory neurons. The addition of the inhibitor of RER-Golgi-TGN transport Brefeldin A to pleural-pedal ganglia after the 5-HT treatment blocked the increase of ApGT1 in synaptosomes observed 24 h after 5-HT (Fig. 7). Brefeldin A blocks RER to Golgi transport and leads to morphological changes due to tubulation and mixing of the TGN and endosomal system. It is unlikely that ApGT1 would be targeted to the endosomal pathway. Moreover, trafficking to and from the plasma membrane appears to remain normal when Brefeldin is present. (Lippincott-Schwartz et al., 1991; Klausner et al., 1992). It seems most likely that Brefeldin A blocked the increase in ApGT1 by disruption somewhere in the RER-Golgi-TGN pathway but disruption of downstream targets cannot be ruled out. Thus, blocking the increase in ApGT1 by Brefeldin A indicates the involvement of RER-Golgi-TGN trafficking. Alternatively, Brefeldin A could be blocking trafficking of proteins important for the induction pathway of the increase in ApGT1 and glutamate uptake by 5-HT and thus, blocking the increase of ApGT1 indirectly. The majority of ApGT1 in pleural ganglia appears to be in a mature form (Endo-H insensitive), suggesting that the pool of ApGT1 might be located past the RER, presumably in the Golgi or TGN as has been observed for other amino acid transporters (Hatanaka et al., 2006).

Under basal conditions, ApGT1 exists as a set of proteins with extensive heterogeneity of charge and small heterogeneity of molecular weight. 5-HT treatment of ganglia changed the pattern of ApGT1 proteins showing that 5-HT regulated the post-translational modifications of ApGT1. The 0.1 pH differences in protein spots observed in 2D gels are consistent with phosphorylation isomers. Furthermore, the number of protein spots in 2D gels is similar to the number of phosphorylation consensus sites for PKA and PKC in the ApGT1 protein sequence. 5-HT activates both PKC and PKA (reviewed in Kandel, 2001), but only PKA is required for the long-term increase in glutamate uptake (Khabour et al., 2004). Thus, it is probable that ApGT1 is phosphorylated, at least by PKA, during 5-HT treatment. Surface expression of mammalian glutamate transporters EAAC1 and GLT1 is modulated by direct phosphorylation of transporters (Casado et al., 1993; Huang et al., 2006). Also, because the carbohydrates may have charged oligosaccharides, ApGT1 may also exhibit 5-HT-induced changes in carbohydrates during maturation as ApGT1 moves through the Golgi.

How could changes in post-translational modifications affect ApGT1 trafficking and surface expression in synaptic terminals? One possibility is that post-translational changes of ApGT1 might facilitate interaction of ApGT1 with glutamate transporter associated proteins, leading to their traffic to the terminals as shown for mammalian glutamate transporters (for review see Gonzalez and Robinson, 2004b, Ruggiero et al., 2008). In agreement with this hypothesis, an increase in labeled co-immunoprecipitated ACOP was induced by 5-HT, suggesting that the interaction between ACOP and ApGT1 might be regulated and important for the increase in ApGT1 by 5-HT. ACOP does not appear to be transported via microtubules to the terminals (see Fig. 4a), suggesting that ACOP might be a cytoplasmic protein. At this point, the nature and source of ACOP remains an intriguing mystery.

Regulation of trafficking (transport to the membrane) is an important mechanism for synaptic plasticity in mammals (for review see Malenka, 2003) and some evidence suggests that regulation of trafficking is also an important mechanism for learning and memory in Aplysia. The trafficking (both endocytosis and exocytosis) of an Aplysia tyrosine kinase receptor-like receptor (ApTrkl) was shown to be regulated by 5-HT (Nagakura et al., 2006). Additionally, endocytosis of a membrane protein, ApCAM, was increased by 5-HT as was the synthesis of clathrin (Bailey et al., 1992; Hu et al., 1993). The trafficking and clustering of AMPA receptors in motor neurons appears to be modulated by synapse formation and 5-HT (Li et al., 2006). Importantly, kinesin, the motor protein involved in anterograde transport, may be regulated by 5-HT (Puthanveettil and Kandel, 2006) and disruption of axonal transport by nocodazole applied to isolated siphon nerves completely blocked LTF at distal synapses (Guan and Clark, 2006), suggesting that transport of proteins may be upregulated and is required during synaptic plasticity.

A constellation of changes are involved in the formation of long-term memories. Our studies in Aplysia, have allowed us to examine one of those changes (glutamate uptake) and compare it with other changes involved in LTS/LTF. LTS/LTF and the increase in glutamate uptake appear to be co-regulated in that the same signaling pathways are involved from training until induction of C/EBP (Kandel, 2001; Khabour et al., 2004; Collado et al., 2005). Our studies suggest that regulation of trafficking of ApGT1 from the Golgi-TGN leading to increased ApGT1 in terminal membranes might be the mechanism of expression of the long-term increase in glutamate uptake. A long half-life of ApGT1 may account at least for the 24h - 48h change in glutamate uptake. Our current hypothesis is that the exit of ApGT1 from the Golgi-TGN is regulated through phosphorylation of ApGT1 by some of the kinases activated during induction of LTF/LTS, leading to increased interaction of ApGT1 with a chaperone protein whose transcription and/or translation is upregulated during LTS/LTF. The study of the regulatory mechanisms of ApGT1 will help shed light on the mechanisms that link induction and expression of memory.

Supplementary Material

Supp Fig S2

Acknowledgements

The authors would like to thank Dr. Oliver Rawashdeh for his help in the development of the Immunoprecipitation technique, Marta Nunez-Regueiro for her help in the 2D gel experiments, and Charity L. Green and Crystal Malone for technical assistance. The authors would also like to thank Dr. Lisa Lyons for helpful comments on the manuscript. This work was supported by NIH grants NS01985 to JHB and NS28462 to AE.

Abbreviations

ApGT1
Aplysia glutamate transporter 1
LTF
long-term facilitation
LTS
long-term sensitization
5-HT
5-hydroxytryptamine (serotonin)

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