Metabolic studies of synaptamide in an immortalized dopaminergic cell line

Prostaglandins and Other Lipid Mediators 141 (2019) 25–33

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Original Research Article

Metabolic studies of synaptamide in an immortalized dopaminergic cell line Shilpa Sonti

⁎,1

T

, Mansi Tolia, Richard I. Duclos Jr., Ralph H. Loring, Samuel J. Gatley

Department of Pharmaceutical Sciences, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, United States

A R T I C LE I N FO

A B S T R A C T

Keywords: Docosahexaenoic acid Fatty acid amide hydrolase Synaptamide N27 cells PF3845

Introduction: Synaptamide, the N-acylethanolamine of docosahexaenoic acid (DHA), is structurally similar to the endocannabinoid N-arachidonoylethanolamine, anandamide. It is an endogenous ligand at the orphan G-protein coupled receptor 110 (GPR110; ADGRF1), and induces neuritogenesis and synaptogenesis in hippocampal and cortical neurons, as well as neuronal differentiation in neural stem cells. Purpose: Our goal was to characterize the metabolic fate (synthesis and metabolism) of synaptamide in a dopaminergic cell line using immortalized fetal mesencephalic cells (N27 cells). Both undifferentiated and differentiating N27 cells were used in this study in an effort to understand synaptamide synthesis and metabolism in developing and adult cells. Methods: Radiotracer uptake and hydrolysis assays were conducted in N27 cells incubated with [1-14C]DHA or with one of two radioisotopomers of synaptamide: [α,β-14C2]synaptamide and [1-14C-DHA]synaptamide. Results: Neither differentiated nor undifferentiated N27 cells synthesized synaptamide from radioactive DHA, but both rapidly incorporated radioactivity from exogenous synaptamide into membrane phospholipids, regardless of which isotopomer was used. Pharmacological inhibition of fatty acid amide hydrolase (FAAH) reduced formation of labeled phospholipids in undifferentiated but not differentiated cells. Conclusions: In undifferentiated cells, synaptamide uptake and metabolism is driven by its enzymatic hydrolysis (fatty acid amide hydrolase; FAAH), but in differentiating cells, the process seems to be FAAH independent. We conclude that differentiated and undifferentiated N27 cells utilize synaptamide via different mechanisms. This observation could be extrapolated to how different mechanisms may be in place for synaptamide uptake and metabolism in developing and adult dopaminergic cells.

1. Introduction Synaptamide (N-docosahexaenoylethanolamine) is an endocannabinoid-like molecule that incorporates docosahexaenoic acid (DHA, C22:6), an omega-3 fatty acid that is a common food supplement. Although evidence for an N-acylethanolamine metabolite of DHA has been documented previously [1] its bioactive nature came into light only recently. Dietary supplementation with DHA itself has been reported to enhance cognition [2,3] and to facilitate neuronal differentiation [4,5], survival [6,7] and synaptogenesis [8,9]. Kim and coworkers, using primary hippocampal and cortical neurons, concluded that the functional effects of DHA in these cultures may be mediated by its conversion into synaptamide [10,11]. In a series of reports, these authors have examined signalling pathways for synaptamide [11–13],

and identified the orphan G-protein coupled receptor 110 (GPR110; ADGRF1), as a synaptamide receptor [14]. Less attention has been given to the synthesis and metabolism of synaptamide than to its bioactive effects. We previously reported the synthesis of two radioisotopomers of synaptamide, labeled with carbon14 on the ethanolamine moiety [α,β-14C2]synaptamide or on the DHA moiety [1-14C-DHA]synaptamide [15]. These two radioisotopomers were utilized as tools to evaluate the brain uptake of exogenous synaptamide and its metabolic fate, similar to our prior work with other N-acylethanolamines, including the endocannabinoid anandamide, the N-acylethanolamine of arachidonic acid [16]. In the present study we investigated the biosynthesis and metabolic pathways of synaptamide in a dopaminergic cell line. Shortly following the discovery of anandamide and its ability to

Abbreviations: DHA, docosahexaenoic acid; AA, arachidonic acid; FAAH, fatty acid amide hydrolase; [αβ-14C2], synaptamidesynaptamide labeled on the ethanolamide moiety; [αβ-14C2], anandamide anandamide labeled on the ethanolamide moiety; [1-14C-DHA], synaptamide synaptamide labeled on the docosahexaenoyl moiety; [1-14C-AA], anandamide anandamide labeled on the arachidonoyl moiety; N27 cells, 1RB3AN27 fetal mesencephalic dopaminergic cells ⁎ Corresponding author at: Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, 02115, United States. E-mail addresses: [email protected], [email protected] (S. Sonti). 1 Present address: 650 W 168th Street, BB 1206; Columbia University, New York, NY 10032. https://doi.org/10.1016/j.prostaglandins.2019.02.002 Received 3 August 2018; Received in revised form 30 January 2019; Accepted 5 February 2019 Available online 11 February 2019 1098-8823/ © 2019 Published by Elsevier Inc.

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dehydroepiandosterone, α-tocopherol, TRIS, bovine serum albumin were obtained from Sigma-Aldrich. The FAAH inhibitor PF3845 was procured from Pfizer [26]. Solvents chloroform, acetone and methanol were obtained from Fisher scientific. Authentic phospholipid standards: 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (856705 P), L-α-phosphatidylinositol (840044 P), 1-palmitoyl-2-hydroxy-sn-glycero3-phosphocholine (855675 P), 1,2-dipalmitoyl-sn-glycero-3-phospho-Lserine (840037 P), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (850705 P) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (850355 P) were purchased from Avanti polar lipids. Solvable and Ultima Gold™ XR, high flash-point liquid scintillation counter cocktail were obtained from Perkin Elmer Labs Inc. Non-radioactive synaptamide was synthesized as previously described [15] and purity was confirmed by NMR.

bind to cannabinoid receptors [17], Devane and Axelrod documented the synthesis of several N-acylethanolamines, including synaptamide, in brain homogenates incubated with C-14 labeled ethanolamine [18]. Subsequent studies confirmed the biosynthesis of synaptamide in hippocampal neurons [8,10], cortical neurons [11] and neural stem cells [13], but did not elaborate on the mechanism of its synthesis. Recent findings of decreased N-acylethanolamine levels in the brains of Nacylphosphatidylethanolamine specific phospholipase D (NAPE-PLD) knock-out mice suggest that synaptamide, like its structural analog anandamide, is synthesized from its N-acylphosphatidylethanolamine precursor [19]. There is no information yet regarding the site of synaptamide synthesis – whether it takes place in neurons or glia—or on the conditions required for its synthesis. Also, there has been no previous investigation of synaptamide uptake and metabolism in vitro. Given the importance of DHA in dopaminergic neuronal survival and differentiation [20,21], we undertook the investigation of synaptamide synthesis, uptake and metabolism in dopaminergic cells. The 1RB3AN27 (N27) cell line is an immortalized fetal rat mesencephalic cell line that produces dopamine and expresses tyrosine hydroxylase and the dopamine transporter. It was created by transfecting fetal rat mesencephalon with a plasmid vector carrying LTa gene from SV40 virus [22]. We employed these cells, which are free of glial cells, in our studies as previous studies on synaptamide were carried in primary hippocampal and cortical cultures [23] but not in dopaminergic cells. As N27 cells were derived from fetal cells, the undifferentiated cells resemble the fetal “dividing” cells and take on the properties of an adult neural cell with the onset of differentiation [24], thus providing as an ideal platform to study the metabolism of synaptamide in developing as well as adult dopaminergic cells. Owing to the structural similarity of synaptamide to anandamide, it is believed that net transport of synaptamide and anandamide into cells involves passive diffusion followed by hydrolysis by FAAH (Fatty Acid Amide Hydrolase). However, the substrate preference of FAAH to Nacylethanolamines with very long acyl chains (> C20) has not yet been demonstrated. One study indirectly looked at FAAH’s substrate preference towards synaptamide [25]; nevertheless no definitive studies appear to have been conducted. Since evidence in support of this assumption was lacking we investigated the role of FAAH on synaptamide metabolism and uptake in dopaminergic cells. Thus, in the present study we hypothesized that synaptamide is synthesized in dopaminergic cells and externally administered synaptamide is taken up into dopaminergic cells through its enzymatic hydrolysis by FAAH. To test this hypothesis, we differentiated N27 cells into neuron-like cells and by using both undifferentiated and differentiating cells, we expected to gain insight into the metabolic fate of synaptamide in developing as well as mature, adult dopaminergic neural cells. To our knowledge, we are the first to characterize the biochemistry of carbon-14 labeled synaptamide in a dopaminergic cell line. The mechanism(s) mediating the bioactive properties of synaptamide is poorly understood. This lack of knowledge hinders the development of pharmacological interventions which may prove to be effective in overcoming neurological deficits. In order to be able to elucidate its biochemical pathways, it is first important to understand the fate of synaptamide in the neural cell. The proposed research is significant because once the metabolic fate of synaptamide is known, it will facilitate the development of testable hypotheses, finally leading to the elucidation of its biochemistry.

2.2. Radiotracers [14C]Docosahexaenoic acid ([14C]DHA), [14C]arachidonic acid ([ C]AA), [14C]anandamide and [14C]ethanolamine were purchased from American Radiolabeled Chemicals or from Moravek as ethanolic solutions. [α,β-14C2]synaptamide and [1-14C-DHA]synaptamide were prepared in house under argon by standard methodology using 1-ethyl3-(3-dimethylaminopropyl)-carbodiimide, followed by purification on silica gel [15]. Stock concentrations of radioactive compounds were formulated in ethanol and subsequent dilution into working concentrations was performed in complete growth medium. The purity of the radiotracers used was assessed prior to each experiment using thin layer chromatography (TLC) and only those fractions which were at least 95% pure were utilized. 14

2.3. N27 cell culture, differentiation and radiotracer addition N27 cells (obtained from Dr. Freed’s lab, University of Colorado) were grown at 37 °C in 5% CO2 in RPMI 1640 with L-glutamine supplemented with 10% Fetal bovine Serum (FBS) and 1% penicillinstreptomycin. Cells (between 8–25 passages) were grown in 75 cm2 (T75) cell culture flasks and for passaging, the confluent cells are separated using 1.4 ml of 0.25% trypsin with 0.1% EDTA. The cells were resuspended in fresh medium and seeded in 60 mm tissue culture dishes at a density of 1 × 106 cells per dish in 3 ml complete growth medium. 24 h following seeding, the medium was replaced by 1X HBSS with 0.1% BSA and the cells were used for uptake experiments. Cells were also plated in a 24 well plate at a density of 5 × 105 cells per well containing one 13 mm round German glass coverslip coated with 100 μg/ml Poly-D-lysine solution in water for immunostaining experiments. The protocol for N27 cell differentiation was obtained from Dr. Freed’s lab [27]. Briefly, 24 h after seeding, differentiating agents – Dibutyryladenosine 3′,5′-byclic monophosphate (dibutyryl cyclic AMP; 2 mM) and dehydroepiandosterone (60 μg/ml), were added to N27 cells in fresh medium and allowed to differentiate for 48 h following which, the cells were either fixed for immunostaining (Fig. 1) or the medium was replaced with 1X HBSS with 0.1% BSA for uptake experiments

2. Materials and methods 2.1. Materials Reagents for culture, RPMI 1640 (with L-Glutamine), 0.25% Trypsin with 0.1% EDTA, Fetal Bovine Serum, penicillin-streptomycin were obtained from Invitrogen (ThermoFisher Scientific). Dibutyryladenosine 3′,5′-byclic monophosphate sodium salt,

Fig. 1. Representative images of undifferentiated (left) and differentiated (right) N27 cells. 26

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or [14C]synaptamide were used.

based on the Oddi et al’s [28] modification of the method established by Fowler et al [29]. Fixed cells were stained for tyrosine hydroxylase using rabbit-anti-TH antibody (1:4000; AB192; Chemicon) to confirm the dopaminergic nature of the cell. Tyrosine hydroxylase immunoreactivity was visualized by using biotinylated goat anti-rabbit secondary IgG antibody (1:250: #BA-1000: Vector Labs) which after conjugation with Vector ABC reagent (to add avidin-HRP to biotin tag) develops a brown color on addition of Vector DAB solution. For uptake experiments, undifferentiated (plated 24 h prior) or differentiating cells (plated 72 h prior and differentiated 48 h prior) in 1X HBSS with 0.1% BSA were placed in a water bath maintained at 37 °C. After 10 min of equilibration at 37 °C, 200 nM radiotracer was added to the cells and incubated for 1, 2.5, 5 or 10 min. After the designated time, the reaction was stopped by aspirating the HBSS into a scintillation vial and the cells were scraped and pelleted in ice cold PBS with 1% BSA. To account for non-specific binding to culture plates, we performed control incubation in parallel with cells on ice at all time points. In all cases, > 90% of the radioactivity added to the incubations was recovered. Cell pellets were harvested and lipids were extracted in chloroform. Hydrolysis of [α,β-14C2]anandamide and [α,β-14C2]synaptamide by the membrane enzymes is expected to release [14C2] ethanolamine which is water soluble and thus partitions into the aqueous phase and can be easily quantified. The percentage of lipid uptake by the cells was determined by subtracting the cell radiolabel uptake in control incubation from their uptake at 37 °C. For FAAH inhibition studies, the cells (both undifferentiated and differentiating cells) were incubated with 0.1% DMSO or 2 μM PF3845 in 0.1% DMSO for 30 min at 37 °C prior to incubation with the respective radiotracers. Data from three experiments for each condition was used for analysis.

2.5.2. Two dimensional TLC analysis Chloroform extracts from cells were spotted on 10 X 10 cm silica gel 60 G F254 plates which were developed in the first dimension for about 45 min in a mobile phase containing chloroform–methanol–ammonia (65:35:5 v/v). The plates were allowed to dry thoroughly, turned at right angles and then developed in the second dimension for another 45 min in a mobile phase containing chloroform-acetone-methanolacetic acid-water (30:40:10:10:5). Authentic non-radioactive phospholipid samples were used as standards. Following development, the TLC plates were air dried and then opposed to phosphor screens to produce autoradiographs. To confirm the identities of radioactive spots, the TLC plates were either charred (to visualize all standards and metabolites – radioactive and nonradioactive) [31] or sprayed with ninhydrin to visualize the phospholipids with a free amino group. Iodine was also used to identify unsaturated compounds. 2.6. Determination of synaptamide hydrolysis Male Swiss-Webster mice were purchased from Taconic Farms. Experimental protocols were approved by Northeastern University Institutional Animal Care and Use Committee (IACUC), and mice were treated humanely in compliance with NIH guidelines for the use of laboratory animals. They were housed in groups of five in disposable plastic cages in the animal facility until the day of use. Mice were euthanized by cervical dislocation and their brains were removed immediately. The whole brain was homogenized using Tris Magnesium EDTA (TME) buffer with 2.5% Bovine Serum Albumin (BSA) to make a final brain homogenate of various concentrations – 20 mg/ml, 10 mg/ ml and 1.25 mg/ml. A portion of the brain homogenate prepared was pre-incubated with the selective and irreversible FAAH inhibitor, PF3845 (0.2 mM) for 30 min at room temperature to validate that these results are due to hydrolysis by FAAH. 1 μl of the prepared stock of [α,β-14C2]anandamide or [[α,β-14C2]synaptamide (0.1μCi/100 μl) was added to the 200 μl of brain homogenates (of each concentration) and incubated in the water bath at 37 °C for 0, 15 or 30 min. This was performed in triplicate. The incubations were retrieved from the water bath and the reaction stopped by the addition of a 1 ml mixture of ice cold chloroform and methanol (1:1) plus 250 μl 2 N Hydrochloric acid. The hydrolysis product, [14C]ethanolamine is an amine base, hence it partitions into the acid layer. The radioactivity of the acid layer therefore reflects the extent of hydrolysis. After 30 min, all samples were centrifuged for 12 min at 4°C and at 14,000 rpm. Following centrifugation, 200 μl of the upper, aqueous layer was pipetted into liquid scintillation vials to which the Ultima Gold™ XR scintillation cocktail was added and radioactivity measured using a liquid scintillation counter.

2.4. Lipid extraction from N27 cells Lipid extractions were obtained from cell pellets of undifferentiated and differentiating cells following the procedure used by Folch et al. [30]. Cell pellets were extracted in 200 μl of the extraction mixture (chloroform/methanol, 2:1) after sonicating on ice twice (30 s each time) and centrifuging at 14,000 rpm for 15 min. The supernatant was transferred to a tube with 0.9% NaCl (40 μl). 100 μl of chloroform was added to cell debris for sonication and the suspension was centrifuged again for 15 min at 14,000 rpm. The supernatants were mixed, vortexed and centrifuged again to separate the organic and aqueous layers, which were transferred into separate tubes. The aqueous layer and an aliquot of the organic (chloroform) layer were used for scintillation counting and the rest of the chloroform layer was stored at −80 °C until TLC analysis. 2.5. Radio-TLC analysis Chloroform extracts were dried under argon and then re-dissolved in 20 μl of chloroform. 1 μl of the extract was used to determine the total amount of radioactivity. To be able to visualize the metabolites efficiently with only a few days’ exposure, a fraction of the extract containing at least a 1000 CPM radioactivity had to be spotted on the silica gel plate. To avoid overloading the plates, the minimum amount of extract containing at least 1000 cpm was spotted. Two types of analyses were carried out – one dimensional TLC to separate the major lipid classes in the cell or brain extracts, and two dimensional TLC to identify specific phospholipids.

3. Results 3.1. Synaptamide synthesis is not observed in N27 cells N27 cells started to differentiate within 48 h in the presence of dibutyryl cyclic AMP (2 mM) and dehydroepiandosterone (60 μg/ml) (Fig. 1). Significant differences in radioactive counts were observed between undifferentiated and differentiating N27 cells incubated with [1-14C]DHA at 10 min time point (p < 0.05) (Fig. 2a). The time dependent increase in radioactive counts in lipid extracts which could represent free fatty acids or metabolites synthesized from radiolabelled fatty acid. To identify the chemical nature of the radioactivity, the lipid extracts (of undifferentiated as well as differentiating cells) were subjected to one dimensional TLC and subsequent autoradiography analysis. Air-oxidation of the radiolabeled standard as it runs along the silica plate or as it dries out cannot be ruled out and manifests as

2.5.1. One dimensional TLC analysis Chloroform extracts from cells were spotted on a 20 X 10 cm silica gel 60 G F254 plates which were developed for about 150 min, using a mobile phase containing chloroform–methanol–ammonia (60:30:1 v/ v). Fatty acid and fatty acid ethanolamide standards were used to identify corresponding spots from reactions in which [14C]anandamide 27

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Fig. 2. (A) Radioactivity in metabolites present in lipid extracts of N27 cells incubated with 200 nM [1-14C]DHA was measured by scintillation counting. Data is expressed in terms of % radioactivity in lipid extracts. N = 3; error bars represent SD. DHA, Docosahexaenoic acid. Statistical analysis is performed using Student’s ttest. *p < 0.05. (B) Representative TLC analysis showing the presence of radioactive metabolites in lipid extracts of undifferentiated (left) and differentiating (right) N27 cells incubated with 200 nM [1-14C]DHA. (C) Representative autoradiograph (right) and charred image of TLC analysis (left) of undifferentiated N27 cells incubated with 200 nM [1-14C]DHA. Charred plates displayed non-radioactive synaptamide (1) and various lipid classes DHA incorporates into: phospholipids (2, 3, and 4) and other triglycerides (top). The autoradiograph displays lipid classes that incorporated [1-14C]DHA. A, Non-radioactive synaptamide; B, Radioactive lipid extract from N27 cells treated with [1-14C]synaptamide; C, mixture of radioactive cell lipid extract and non-radioactive synaptamide.

“smudgy” appearance on the autoradiographs just below the actual hot spots. TLC analysis confirmed the presence of various metabolites in the lipid extracts; however, the presence of synaptamide among the metabolites is not clear as none of the radioactive hot spots from the autoradiographs align with that of [1-14C-DHA]synaptamide standard (Fig. 2b). In order to determine the presence of [1-14C-DHA]synaptamide in lipid extracts of cells incubated with [1-14C]DHA, one dimensional TLC analysis was performed with the lipid extract co-spotted with non-radioactive synaptamide standard. The developed TLC plates were charred after being treated with copper sulfate/phosphoric acid reagent. Comparison of charred plates with autoradiographs recorded prior to charring revealed no overlap in the synaptamide spots from standards and lipid extracts (Fig. 2c) indicating the absence of synaptamide in cells incubated with [1-14C]DHA.

including anandamide (mediated by FAAH) drives further uptake into the cell. Since a similar pattern was observed with synaptamide, its intracellular metabolism was also expected to be involved in its uptake into undifferentiated N27 cells. To determine if hydrolysis by FAAH drives synaptamide uptake, undifferentiated and differentiating N27 cells were incubated with a specific FAAH inhibitor, PF3845 to eliminate FAAH activity prior to analyzing [α,β-14C2]synaptamide uptake. FAAH inhibition resulted in a significant decrease in radioactive counts in both lipid and aqueous extracts in undifferentiated cells suggesting that hydrolysis by FAAH is the major driving force for its uptake, analogous to uptake of radiolabeled anandamide with and without the presence of PF3845 (Table 1). In contrast, FAAH inhibition did not seem to have an effect on either [α,β-14C2]synaptamide uptake or hydrolysis in differentiating cells (Table 1). To confirm this, different concentrations of mouse brain homogenates (1.25, 10 and 20 mg/ml) were used to perform FAAH activity assays. The extent of synaptamide hydrolysis was directly proportional to time and tissue concentration. Inhibition of FAAH with 2 μM PF3845 (Ki for FAAH = 230 nM) significantly decreased [α,β-14C2]synaptamide hydrolysis only at the highest tissue concentration (20 mg/ml) at the longest time point (30 min) of incubation (Fig. 3a); while hydrolysis of [α,β-14C2]anandamide was significantly inhibited in all conditions (Fig. 3b).

3.2. Synaptamide uptake in N27 cells is driven by its hydrolysis Undifferentiated N27 cells incubated with [α,β-14C2]synaptamide revealed a time dependent increase in radioactive counts in both lipid and aqueous extracts which is representative of increased synaptamide uptake and hydrolysis respectively. Interestingly, this time dependent accumulation of radioactivity was not observed in the lipid extracts of differentiating cells. This pattern is similar to, but slower than that observed with [α,β-14C2]anandamide, suggesting that N27 cells have a preference towards anandamide over synaptamide (Table 1). The metabolic removal of intracellular N-acylethanolamines, 28

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Table 1 Comparison of % Radioactivity in lipid (chloroform) and aqueous extracts of undifferentiated and differentiating N27 cells following incubation with 200 nM [α,β-14C2]anandamide or [α,β-14C2]synaptamide with or without the FAAH inhibitor, PF3845. Data is expressed as mean ± SD; N = 3; Statistical analysis performed using Student’s t-test. UNDIFFERENTIATED CELLS TIME (MIN)

% RADIOACTIVITY IN LIPID EXTRACT 14

1 2.5 5 10 1 2.5 5 10

[α,β- C2] anandamide 0.23 ± 0.19 0.42 ± 0.37 1.11 ± 0.54 1.68 ± 0.78 [α,β-14C2] synaptamide 0.14 ± 0.15 0.16 ± 0.24 $ 0.31 ± 0.04 0.72 ± 0.03

% RADIOACTIVITY IN AQUEOUS EXTRACT -14

[α,β C2] anandamide + PF3845 0.16 ± 0.11 0.14 ± 0.03 * 0.14 ± 0.05 * 0.19 ± 0.08 [α,β-14C2] synaptamide + PF3845 0.05 ± 0.06 0.15 ± 0.1 0.23 ± 0.1 * 0.34 ± 0.1

[α,β-14C2] anandamide 0.02 ± 0.03 0.12 ± 0.09 0.21 ± 0.11 0.19 ± 0.06 [α,β-14C2] synaptamide 0.01 ± 0.01 0.01 ± 0.002 $ 0.03 ± 0.02 $ 0.06 ± 0.02

[α,β-14C2] anandamide + PF3845 0.001 ± 0.001 0.001 ± 0.001 * 0.004 ± 0.005 * 0.007 ± 0.009 [α,β-14C2] synaptamide + PF3845 0.001 ± 0.001 0.004 ± 0.006 * 0.003 ± 0.003 * 0.008 ± 0.006

DIFFERENTIATING CELLS TIME (MIN)

1 2.5 5 10 1 2.5 5 10

% RADIOACTIVITY IN LIPID EXTRACT

% RADIOACTIVITY IN AQUEOUS EXTRACT

[α,β-14C2] anandamide 0.07 ± 0.05 0.16 ± 0.1 # 0.08 ± 0.03 #0.21 ± 0.09 [α,β-14C2] synaptamide 0.01 ± 0.01 0.17 ± 0.01 # 0.06 ± 0.02 $,# 0.04 ± 0.01

[α,β-14C2] anandamide 0.04 ± 0.03 0.09 ± 0.04 0.15 ± 0.05 0.23 ± 0.15 [α,β-14C2] synaptamide 0.001 ± 0.0001 $ 0.001 ± 0.008 $ 0.006 ± 0.006 $ 0.006 ± 0.001

[α,β-14C2] anandamide + PF3845 0.11 ± 0.05 0.12 ± 0.06 0.22 ± 0.17 0.21 ± 0.1 [α,β-14C2] synaptamide + PF3845 0.001 ± 0.001 0.01 ± 0.01 #0.05 ± 0.01 #0.06 ± 0.03

[α,β-14C2] anandamide + PF3845 0.06 ± 0.06 0.06 ± 0.07 0.15 ± 0.02 0.11 ± 0.1 [α,β-14C2] synaptamide + PF3845 0.001 ± 0.001 0.001 ± 0.0006 * 0.006 ± 0.003 * 0.006 ± 0.004

* p < 0.05 difference between the % radioactivity in extracts of cells pretreated with versus without the FAAH inhibitor (PF3845) before incubating with either ethanolamide. # p < 0.05 difference between the % radioactivity in extracts of undifferentiated versus differentiating cells treated with either ethanolamide with or without PF3845. $ p < 0.05 difference between the % radioactivity in extracts of cells treated with [α,β-14C2]anandamide versus [α,β-14C2]synaptamide (either with or without PF3845).

did not reveal color in other phospholipid spots, it could be hypothesized as either phosphatidylserine [12], phosphatidylcholine (PC) or Ndocosahexaenoyl phosphoethanolamines (NAPE). The RF values of PC and PS in one dimensional TLC are very close making it difficult to interpret the identity of the spot. A two dimensional TLC analysis identified the second spot as phosphatidylcholine (Fig. 4d).

3.3. Synaptamide partitions into phospholipids in vitro Free fatty acids liberated from fatty acid ethanolamides in in vitro systems are promptly incorporated into various membrane lipids [32,33]. Results from our study also substantiate this observation. TLC analysis of lipid extracts from undifferentiated as well as differentiating N27 cells treated with [α,β-14C2]synaptamide shows that along with corresponding N-acylethanolamines, the radiolabel is incorporated into several polar lipids: phospholipids (Fig. 4a). Supporting our observation that FAAH is responsible for synaptamide hydrolysis, TLC analysis of cell extracts pretreated with PF3845 show that FAAH inhibition significantly decreased the label incorporation into phospholipids and increased the level of intact N-acylethanolamines in cells (Fig. 4b). As [α,β-14C2]synaptamide is labeled on the ethanolamine moiety, it is reasonable to expect that the labeled ethanolamine released upon [α,β-14C2]synaptamide hydrolysis is incorporated into phospholipids incorporating the [14C]ethanolamine: phosphatidylethanolamine (PE) and lyso-phosphatidylethanolamine (LYSO-PE). This was further confirmed when on treatment with ninhydrin one of the spots turned a bright pinkish-purple color, indicative of a compound with free amine (not shown). To resolve the fate of the docosahexaenoyl chain of synaptamide, we used [1-14C-DHA]synaptamide, previously synthesized in our lab, with the radiolabel on its docosahexaenoyl moiety [15]. Lipid extracts of undifferentiated cells treated with either synaptamide radiotracer showed radiolabel incorporation into phospholipids to a similar extent (Fig. 4c); TLC analyses indicated synaptamide partitioned into PE. However, in differentiating cells, the percentage of phospholipid formed was higher when treated with [1-14C-DHA]synaptamide indicating that the docosahexaenoyl chain released from synaptamide partitions into phospholipids other than PE. Since ninhydrin treatment

4. Discussion To our knowledge, ours is the first study to investigate the synthesis, uptake and metabolism of synaptamide in a dopaminergic cell line. N27 cells are particularly interesting because undifferentiated cells are derived from rat fetal mesencephalon and resemble “developing cells” and the differentiated cells undergo morphological changes and resemble “adult neuronal” cells. Using these cells provides a preliminary insight about the importance of synaptamide supplementation in developing as well as mature, adult neural cells. Hence, both undifferentiated and differentiating cells were used in this study. N-acylphosphatidylethanolamines (NAPE’s) are phospholipid precursors for the synthesis of N-acylethanolamines [34] via phospholipase D [35]. Synthesis of N-acylphosphatidylethanolamine is the rate limiting step in any N-acylethanolamine formation and is usually mediated by the membrane associated enzyme, N-acyltransferase (NAT) [36–40]. N-acyltransferase catalyzes the trans-acylation reaction between a donor phospholipid (phosphatidylethanolamine or phosphatidylcholine) and phosphatidylethanolamine. N-acyltransferase activation is triggered in the presence of high levels of intracellular calcium representing the “demand” for N-acylethanolamine synthesis [40]. Owing to the structural similarity between anandamide and synaptamide, the reaction conditions established for anandamide synthesis from 29

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Fig. 3. (A) Time dependent hydrolysis of [α,β-14C2] synaptamide in crude brain homogenates – 1.25 mg/ ml, 10 mg/ml and 20 mg/ml with or without the presence of FAAH inhibitor, PF3845. Data is expressed in terms of % radioactivity quantified in aqueous layers. Error bars represent SD; N = 3. Statistical analysis is performed using Student’s t-test. *p < 0.05. (B) Time dependent hydrolysis of [α,β-14C2]anandamide in crude brain homogenates – 1.25 mg/ml, 10 mg/ml and 20 mg/ml with or without the presence of FAAH inhibitor, PF3845. Data is expressed in terms of % radioactivity quantified in aqueous layers. Error bars represent SD; N = 3. Statistical analysis is performed using Student’s t-test. *p < 0.05.

inhibition by FAAH is only observed at longer time points for synaptamide versus earlier time points for anandamide (Table 1) indicating that FAAH has a greater ability to hydrolyze anandamide than synaptamide. This is further substantiated by carrying out FAAH activity assay by inhibiting FAAH activity using PF3845 in mouse brain homogenates. In addition, compared to anandamide, the cellular synaptamide uptake is slower, suggesting that in undifferentiated N27 cells, hydrolysis driven metabolism of synaptamide is slower than that of anandamide. This supports our observation that FAAH is less effective in hydrolyzing synaptamide than anandamide, but can be responsible for synaptamide hydrolysis either when present in excess or when synaptamide is the only substrate. It is well established that FAAH can hydrolyze a wide range of Nacylethanolamines including anandamide and N-palmitoylethanolamide [47,48]. While anandamide uptake can be regulated by its metabolism by FAAH [49], recent studies show that the activity of FAAH is also influenced by its lipid environment. Anandamide preferentially associates itself to lipid domains of the ER that contain cholesterol. FAAH perceives the presence of this anandamide localized to the ER and preferentially hydrolyses it. This exclusive selectivity is limited to anandamide [50]. The nature of the association of synaptamide with cholesterol is not known yet. Moreover, FAAH preferentially hydrolyzes unsaturated compounds that assume the hair-pin conformation [51] exhibited by anandamide. No information about synaptamide’s conformation is available, but it is possible that the modest effect of FAAH on synaptamide hydrolysis observed in our study reflects a conformation unfavorable for FAAH. In our studies using differentiating N27 cells, FAAH inhibition did not seem to have an effect on either [α,β-14C2]synaptamide uptake or hydrolysis, while the uptake of [α,β-14C2]anandamide even in

arachidonic acid were used to document synaptamide synthesis in N27 cells, but we failed to establish the synthesis of synaptamide in N27 cells, in either undifferentiated or differentiating cells. Possible explanations for this failure include low expression levels in N27 of acylcoenzyme A (acyl-CoA) synthetases with high specificity for DHA relative to AA (e.g. ASCL6). We are unaware of studies that have evaluated the expression of acyl-CoA synthetases in these cells. Another possibility for the lack of synaptamide synthesis is that the incubation time may not be sufficient for its synthesis from [1-14C]DHA as the cells were incubated with the radiotracer for a maximum of 20 min. This is however unlikely as when cells were incubated with exogenous synaptamide, it was rapidly metabolized into phospholipids. Therefore, if the rate of synthesis of synaptamide is much slower than the rate of its metabolism, it might account for why intact synaptamide was not isolated or visualized. Transport of N-acylethanolamines, in particular anandamide, can be viewed as a three step process – adsorption, transmembrane diffusion, and desorption [41]. Enzymatic metabolism of anandamide, inside the cells is expected to be the driving force for its net uptake across the cell membrane [42]. Anandamide is the preferred substrate of fatty acid amide hydrolase (FAAH), a serine hydrolase which is usually associated with the inner cell membrane [43]. Expression of FAAH has been documented in certain cell lines (human U937 leukemia cells [32], striatal and cortical neurons [44]) where anandamide metabolism occurs. As a result its flux into the cells is thought to be mediated via maintenance of a concentration gradient by FAAH activity [29,45]. To date, FAAH expression or FAAH activity has not been documented in N27 cells. FAAH, which is known to hydrolyze other N-acylethanolamines [46] may also be responsible for hydrolyzing synaptamide into DHA and ethanolamine. However, in undifferentiated cells, the 30

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Fig. 4. (A) Quantification of radioactive spots from TLCs shows the partitioning of [α,β-14C2]synaptamide into phospholipids in undifferentiated and differentiating N27 cells. Data is expressed as mean of the % radioactivity ± SD partitioned into phospholipids and the %radioactivity remained as intact [α,β-14C2]synaptamide. Error bars represent SD; N = 4 ([α,β-14C2]synaptamide). (B) Quantification of radioactive spots from TLCs shows the partitioning of [α,β-14C2]synaptamide into phospholipids in undifferentiated and differentiating N27 cells pre-incubated with FAAH inhibitor, PF3845. Data is expressed as mean of the % radioactivity ± SD partitioned into phospholipids and the %radioactivity remained as intact [α,β-14C2]synaptamide. Error bars represent SD; N = 4 ([α,β-14C2]synaptamide). (C) Quantification of radioactive spots from TLCs shows the partitioning of [α,β-14C2]synaptamide and [1-14C-DHA]synaptamide into phospholipids in undifferentiated and differentiating cells. Data is expressed as mean of the % radioactivity ± SD partitioned into phospholipids and the %radioactivity remained as intact labelled synaptamide. Error bars represent SD; N = 3 ([1-14C]synaptamide), N = 4 ([α,β-14C2]synaptamide). (D) Comparison of charred TLCs of standard phosphatidylserine (left), phosphatidylcholine (middle) and the lipid extract of cells incubated with [α,β-14C2]synaptamide (right). The charred image confirms the phospholipid spot in the cell lipid extract. TLC plates were run in two dimensions: dimension 1 – chloroform/methanol/ammonium hydroxide (65/35/5); dimension 2 – chloroform/ acetone/methanol/acetic acid/water (30:40:10:10:5).

expensive alternative to isolating primary mesencephalic neurons from rodent pups. N27 cells have limitations as they are not “true” neuronal cells and the results may vary when studies are carried out in primary cultures. However, they are dopaminergic in nature and hence can be considered a valid model for our studies. b Differentiation of N27 cells is brought about by the differentiating agents, Dibutyryladenosine 3′,5′-byclic monophosphate (dibutyryl cyclic AMP; 2 mM) and dehydroepiandosterone (60 μg/ml). The onset of differentiation resulted in certain amount of cell loss. To compensate for this, the cells to be differentiated were plated 72 h prior and the differentiating agents were added 48 h prior to the addition of the radiotracer. The undifferentiated cells were plated 24 h prior to the addition of the radiotracer, at a similar density as the differentiating cells on the same day. c We used Student's t-test to calculate the p value for most analyses, even where multiple measures are involved. Repeated measures ANOVA would not be an appropriate test because the experimental design of the study is not suitable for this test: the "cell" population had to be different for each measure to enable lipid extraction at each time point.

differentiating cells is driven by its hydrolysis by FAAH. This observation argues against our hypothesis that FAAH mediates synaptamide uptake as we do not see its effect in differentiating cells. We thus believe that N27 cells seem to develop a FAAH independent synaptamide uptake process with the onset of differentiation. Cellular radioactivity derived from labelled synaptamide is almost completely concentrated in the phospholipids phosphatidylethanolamine and phosphatidylcholine [52]. This demonstrates that DHA is released from synaptamide, via hydrolysis by FAAH or other enzyme(s). CDP-ethanolamine:diacylglycerol ethanolamine phosphotransferase, the enzyme involved in the synthesis of phosphatidylethanolamine preferentially uses diacylglycerols rich in DHA [53] explaining the presence of DHA in phosphatidylethanolamine. Since DHA is the preferred fatty acid for phosphatidylserine (PS) synthesis [54], we expected that cell and brain extracts incubated with synaptamide or DHA would result in the formation of phosphatidylserine. 2D-TLC of lipid extracts did not reveal the formation of phosphatidylserine suggesting that longer incubation times are probably required for its synthesis, or that N27 cells lack PSS2 enzymes which preferentially uses DHA-containing phosphatidylethanolamine for the synthesis of phosphatidylserine [55].

6. Conclusion 5. Limitations In conclusion, this study did not support our hypothesis that synaptamide is synthesized in dopaminergic cells, and that externally administered synaptamide is taken up into dopaminergic cells through its enzymatic hydrolysis by FAAH with respect to differentiating N27 cells. We did not observe synaptamide synthesis in N27 cells when using conditions suitable for anandamide synthesis, suggesting that neuronal cell types either do not support synaptamide synthesis, or involve a different mechanism yet to be identified. Further studies are

This study was designed to characterize the biosynthetic pathway of synaptamide, a novel lipid messenger in a dopaminergic cell line; however, it is not without limitations: a We had access to N27 cells which are fetal cells that can be differentiated into adult cells and potentially provide us with two models to study biosynthetic pathway of synaptamide. This was a less 31

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needed to confirm this assumption. Our uptake results show that while the end fate of synaptamide in both undifferentiated (developing) and differentiating (adult) cells is incorporation into phospholipids, the process involved is clearly different. In undifferentiated cells, uptake and metabolism of synaptamide is FAAH dependent, with decreased radiolabel incorporation into phospholipids when FAAH is inhibited. In differentiating cells, uptake, metabolism and incorporation of synaptamide into phospholipids seemed to be independent of FAAH. It can thus be concluded that the fate of synaptamide in N27 cells essentially depends on their differentiation state. Its metabolic pattern in differentiating cells is consistent with previous observations reported in primary hippocampal neurons. Despite the limitations, this study provides valuable insight into the metabolic pathway of synaptamide; however, much remains unclear about the biochemistry and physiology of synaptamide that should be investigated in further studies.

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Declaration of interest Dr. Duclos was supported under a grant to S. J. Gatley from Department of Energy, Office of Science, DER, during the conduct of the study. The other authors have nothing to declare.

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Acknowledgement [22]

This work was supported by the Department of Energy, Office of Science, DER (grant number (DE SC0005251).

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