Characterization of a Fatty Acid Amide Hydrolase (FAAH) in Hirudo verbana

The endocannabinoid system plays a critical role in modulating both peripheral and central nervous system function. Despite being present throughout the animal kingdom, there has been relatively little investigation of the endocannabinoid system beyond the traditional animal model systems. In this study, we report on the identification and characterization of a fatty acid aminohydrolase (FAAH) in the medicinal leech, Hirudo verbana. FAAH is the primary enzyme responsible for metabolizing the endocannabinoid signaling molecule arachidonoyl ethanolamide (anandamide or AEA) and therefore plays a critical role in regulating AEA levels in the nervous system. This Hirudo FAAH (HirFAAH) is expressed in the leech central nervous system (CNS) and is an orthologue of FAAH-2 observed in vertebrates. Functionally, HirFAAH has serine hydrolase activity based on activity-based protein profiling (ABPP) studies using the fluorophosphonate probe TAMRA-FP. HirFAAH also hydrolyzes arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a substrate specific to FAAH. Hydrolase activity during both the ABPP and AAMCA assays was eliminated by mutation at a conserved activity-binding site. Activity was also blocked by the known FAAH inhibitor, URB597. Treatment of Hirudo ganglia with URB597 potentiated synapses made by the pressure-sensitive mechanosensory neuron (P cell), mimicking the effects of exogenously applied AEA. The Hirudo CNS has been a useful system in which to study properties of endocannabinoid modulation of nociception relevant to vertebrates. Therefore, this characterization of HirFAAH is an important contribution to comparative studies of the endocannabinoid system.


Introduction
Endocannabinoids are lipid signaling molecules with a broad range of neuromodulatory effects, especially at the synaptic level [1][2][3].Functionally, endocannabinoids are involved in physiological and behavioral processes that include, neurodevelopment, in ammation, pain, cognition, control of affect, appetite and feeding, seizures, brain injury, and neurodegenerative diseases [4].2-arachydonoyl glycerol (2-AG) and arachidonoyl ethanolamide (anandamide or AEA) are the two most prevalent endocannabinoids.Their actions are mediated by metabotropic CB1 and CB2 receptors, TRPV1 channels, and the orphan G-protein coupled receptor, GPRC55, although other receptors are may also contribute, e.g., peroxisome proliferator antigen receptors (PPAR).Endocannabinoids are unconventional transmitters in that they are not stored in synaptic vesicles, but are instead synthesized "on demand", i.e., in an activity-dependent manner, and often in postsynaptic neurons.2-AG synthesis is mediated diacyl glycerol lipase (DAGL) and is degraded primarily by monoacylglycerol lipase (MAGL), but alternative pathways include alpha/beta hydrolase (ABHD) 6 and 12 [5].AEA synthesis can be mediated by a Narachidonoyl phosphatidyl ethanol phospholipase D (NAPE-PLD) or by several different multi-enzyme processes [6].AEA degradation is mediated by fatty acid aminohydrolase (FAAH) to produce arachidonic acid (AA) and ethanolamine [7], however cyclo-oxygenase 2 (COX-2) has recently been described as an alternative AEA metabolism route [8].
Given the role of endocannabinoids in so many neurophysiological processes, there is considerable interest developing cannabinoid-based therapies for a variety of mental health and neurological conditions [4].However, relatively little progress has been made in translating research of the endocannabinoid system into actual therapies.For example, while there is considerable interest in using either phyto-or endocannabinoid-based therapies to treat chronic pain, there are still no approved treatments, and the International Association for the Study of Pain does not currently endorse cannabinoid-based therapies to treat pain.Greater success in translation of potential cannabinoid-based therapies depends on greater understanding of the basic biology of the endocannabinoid system.This greater understanding would be facilitated by increased use of comparative approaches to identify evolutionarily conserved mechanisms mediating endocannabinoid neurophysiological and neurobehavioral processes.Such comparative approaches have been successful in understanding fundamental processes, such as those related to learning and memory or neurodevelopment [9,10].

Bioinformatics analysis of HirFAAH
A potential sequence for fatty acid amide hydrolase in Hirudo verbana (HirFAAH) accession #GGIQ01042388.1) was identi ed by tBLASTn search of the H. verbana Transcriptome Shotgun Assembly (TSA) sequences using human FAAH (accession NP_001432) as the query sequence.The cDNA sequence was translated using ExPaSy software (https://web.expasy.org/translate/)and the deduced amino acid sequence was analyzed using the protein Basic Local Alignment Search Tool (blastp) from the National Center of Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
The molecular mass of HirFAAH2 was determined using ExPaSy compute tool (https://web.expasy.org/compute_pi/).Comparison of the putative Hirudo FAAH sequence with human FAAH was carried out using the Ident and Sim algorithms from the Sequence Manipulation Suite (https://www.bioinformatics.org/sms2/ident_sim).

Cloning and site mutation of HirFAAH
All primers used for cloning projects are listed in Table 1.The full-length coding sequence of HirFAAH2 was generated from total RNA using SuperScript III One-Step RT-PCR system with Platinum Taq DNA Polymerase (Thermo Fisher Scienti c Inc., Rockford, IL).To generate an expression construct with 3' eGFP tag the HirFAAH was inserted into pcDNA3.1eGFP(Addgene plasmid #13031; http://n2t.net/addgene:13031;RRID:Addgene_13031).Single point mutation was generated using the GeneTailor Site-Directed Mutagenesis System (Thermo Scienti c).All constructs were con rmed by sequencing (Euro ns MWG Operon, Huntsville, AL).Plasmids were transfected into 293HEK cells (ATCC, Manassas, VA) using Lipofectamine 3000 (Thermo Scienti c) as previously described by the manufacturer.For Western blots equal amounts of protein were separated on 10% SDS-PAGE gels and transferred to PVDF.Primary antibodies used were mouse anti-alpha tubulin (Abcam, Cambridge, MA), and mouse anti-GFP (B-2) (Santa Cruz Biotechnology, Dallas, TX).Goat anti-mouse IRDye 680LT was used as a secondary antibody (LiCor Biosciences, Lincoln, NE).Images were taken using the Odyssey CLx and processed using the Image Studio version 5.2 (LiCor Biosciences).Indirect immuno uorescence microscopy 293HEK cells were seeded onto 12 mm coverslips (Carolina Biological, Burlington, NC) and transfected using Lipofectamine 3000 according to the manufacturer's speci cations.48 hours post-transfection the cells were xed with 4% paraformaldehyde and permeabilized with 0.05% Triton X-100.Coverslips were counterstained with DAPI and mounted onto glass slides with ProLong Gold antifade mounting medium (Thermo Scienti c).Slides were viewed on an Olympus BX60 uorescent microscope using the 60X objective and images were captured with a Nikon DS-QilMc Camera.

Activity-based protein pro ling of serine hydrolases
The uorophosphonate probe TAMRA-FP (ActivX Fluorophosphonate Probes, Thermo Scienti c) was used to analyze HirFAAH membrane fractions by activity-based protein pro ling (ABPP) as previously described [20].Brie y, 100 µg of membrane fraction was pretreated with 2% DMSO or the FAAH inhibitor URB597 (Cayman Chemicals, Ann Arbor, MI) for one hour at room temperature.This was followed by incubation with 2 µM TAMRA-FP for three hours at room temperature.To stop the reaction 4X SDSloading buffer was added to a nal concentration of 1X and the proteins were resolved on a 10% SDS-PAGE gel.Fluorescence images of the gels were taken with a Typhoon imager (GE Healthcare, Pittsburg, PA) and analyzed using the Image Studio version 5.2 software (LiCor Biosciences).

Preparation of microsome fractions
FAAH activity was measured in microsomes isolated from HEK cells that over-expressed GFP, HirFAAH or HirFAAH-S225A using a uorogenic substrate assay as previously described [28].Brie y, HEK-293 cells expressing HirFAAH, HirFAAH_S225A or vector alone were grown for 48 hours after transfection.The cells were washed twice with ice-cold DPBS and were spun down by centrifugation at 2000 rpm for 10 min at 4 o C. Cell pellets were snap frozen in liquid nitrogen and stored at -80 o C. The cell pellets were thawed on ice, resuspended in microsome buffer containing 50 mM HEPES (pH 7.4), 1 mM EDTA, and Pierce ™ Protease Inhibitor Mini Tablet, according to manufacturer's recommendations, and were sonicated ve times for 10 sec, resting for 15 sec on ice between each interval.Immediately following sonication, the cell lysate was centrifuged at 12,000 g for 20 min at 4 o C. The pellet was saved as the crude membrane fraction and the supernatant was centrifuged at 100,000 g for 45 min in TLA 100.3 rotor in an Optima Max-XP ultracentrifuge (Beckman).The pellet, microsome fraction, was resuspended in microsome buffer without protease inhibitor cocktail by brief, 10 sec, sonication.Protein concentration was determined with Pierce ™ BCA Assay kit, the samples were aliquoted, snap frozen in liquid nitrogen and stored at -80 o C.

AAMCA hydrolysis by HirFAAH microsomes
The AAMCA (7-amino-4-methyl Coumarin-Arachidonamide) hydrolysis assay was performed in 96-well black plates (Nunc) in a total volume of 100 µl.To begin, 0.5 µg microsomal protein (in 50 µl), prepared as described above, was incubated with or without the designated inhibitor in assay buffer (50 mM HEPES, 1 mM EDTA (pH7.4) and 1.4 mg/ml bovine serum albumin (BSA, 0.1% nal concentration)) for 30 min at room temperature.Immediately following the incubation, 1 µM AAMCA (Cayman Chemical)substrate prepared in assay buffer was added to the microsomal protein, the components were shaken for 2 minutes and measured kinetically for 60 min at 37 o C on a Perkin Elmer VICTOR Nivo (PerkinElmer, Waltham, MA) microplate reader.Pure AMC (7-amino-4-mthylcoumarin, Cayman Chemical) was used to generate 0, 20, 40, 60, 80, and 100 pmol/well standard curve of AMC standard in assay buffer.Values were corrected for background uorescence observed from well containing buffer alone.

Electrophysiology
Electrophysiology experiments were conducted as recently described in Franzen and Burrell (2023).
Brie y, 2-3 g Hirudo verbana (North America BioPharma, Erie, CO) were kept in arti cial pond water (0. 5mM NaOH, 10 mM glucose and 10mM HEPES; pH = 7.4) with at a rate of ≈ 2 ml/min.Individual neurons in a ganglion were viewed via a stereomicroscope under dark eld illumination.Dual intracellular recordings were made from one of the pressure-sensitive mechanosensory neurons (P cells) and one of its postsynaptic targets the motor neuron-like anterior pagoda (AP) neuron.Both the P and AP cells were identi ed by their position and characteristic action potential shape [29].The P-to-AP synapse has been previously identi ed as monosynaptic, glutamatergic synapses [30].Current clamp recordings of P and AP cells were made with a bridge ampli er (BA-1S; NPI, Tamm, Germany) and signals digitally converted for analysis using a DigiData 1322 A (Molecular Devices, Sunnyvale, CA).Current injection into the cells was accomplished using a digital stimulator (Multichannel Systems STG1004; Reutlingen, Germany).
Twin P cell action potentials were elicited with a 300 msec interval.The rst elicited excitatory post synaptic potential (EPSP) was used to measure changes in amplitude between the pre-and post-test.The second EPSP was used to measure the paired-pulse ratio (PPR = 2nd EPSP/1st EPSP) and provide an indication of whether changes in EPSP amplitude have a pre-or postsynaptic loci [31,32].This combined EPSP/PPR recording was repeated at 20 second intervals until 5-10 recordings were obtained and the EPSPs averaged to obtain the rst EPSP amplitude and the second EPSP amplitude to calculate the PPR.
Post-synaptic input resistance (IR) was monitored by delivering a 500ms, 1nA negative current pulse delivered at 20 second intervals (alternated with the EPSP/PPR recordings).The post-synaptic neuron was hyperpolarized to approximately − 70 mV during EPSP and input IR recordings to prevent postsynaptic action potentials which would interfere with EPSP measurements.
Anandamide (AEA), the FAAH inhibitor URB597, and the TRPV inhibitor SB366791 (Tocris) were prepared on the day of the experiment from frozen aliquots (all stock solutions in DMSO).Following pretest EPSP, PPR and IR recordings, drugs were bath-applied via perfusion of the chamber for 15 mins.For vehicle control experiments, ganglia were treated with 0.001% DMSO.This was followed by a 60 min washout period in normal saline and then post-test measurements of the EPSP, PPR and IR.Following the pretest recording, microelectrodes were removed from the P and AP cells to prevent damage due to osmotic stress and re-inserted during the post-test.Percent change of the IR between the pre-and post-test recordings was used to assess the quality of recordings and only experiments < 15% change in input resistance were included for analysis.Changes in synaptic transmission in a given experiment were based on the percent change in the post-test EPSP amplitude relative to the pretest level (i.e., 100* (EPSP post /EPSP pre ).The percent change in PPR between the pre-and post-test records was calculated in the same way.

Statistics
All data are presented as mean ± standard error.Data were analyzed using 2-way and 1-way Analysis of Variance (ANOVA) using Graphpad/Prism.When appropriate Tukey post-hoc comparisons were also carried out.

HirFAAH homology and sequence analysis
Query of the H. verbana TSA using human FAAH amino acid sequence as the query sequence identi ed the transcribed RNA sequence GGIQ01042388.1.Further analysis of GGIQ01042388.1 resulted in the predicted HirFAAH with a nucleotide length of 1,578 bp, an amino acid length of 525 amino acids and estimated mass of 59.3 kDa.The predicted HirFAAH appears to be more similar to human FAAH-2 (HsaFAAH-2) isoform, based on a 44.1% sequence identity and 59.04% similarity (Fig. 1).The HirFAAH sequence compared to human FAAH-1 revealed 19.19% sequence identity and 33.39% similarity.Consistent with FAAH is other species the HirFAAH sequence possesses a GXSXG motif, a conserved feature in serine hydrolases (black box, Fig. 1).In addition, HirFAAH possesses an amidase signature sequence that exhibits 60.3% sequence identity and 71.1% similarity to HasFAAH2.This amidase sequence contained the serine-serine-lysine catalytic triad (grey highlight and bold/underlined residues, respectively, Fig. 1) and putative catalytic serine (S225) which aligns with the catalytic serine (S230) of HsaFAAH-2 (black arrow, Fig. 2) and are critical for FAAH serine hydrolase activity [33,34].Using these sequence data, subsequent qRT-PCR experiments con rmed the expression of HirFAAH in the Hirudo central nervous system (CNS; Fig. 2).

Features of HirFAAH expression in HEK-293 cells
The cloned HirFAAH was labeled in frame with a C-terminal eGFP tag resulting in a protein 768 amino acids in length and expressed in 293-HEK cells (Fig. 4).Immuno uorescence microscopy indicated the tagged HirFAAH was localized to hollow, ring-like cytoplasmic structures (Fig. 4a, b).This is consistent with immuno uorescence microscopy experiments performed with HsaFAAH2 that also localized to ringlike structures (Fig. 4a, b) [35].The eGFP tagged HirFAAH protein was enriched in the membrane fractions of transfected 293-HEK cells (Fig. 4B, C), again consistent with the subcellular distribution of other FAAHs [36,37].We also tested a version of HirFAAH with a mutation of the active site serine (S225A).This mutation did not prevent expression of HirFAAH in the 293-HEK cells, nor did it disrupt localization to the membrane fraction as seen in wildtype FAAH (Fig. 4c, d).

Activity-Based Protein Pro ling
The catalytic activity of HirFAAH was assessed by ABPP, which detects functionally active serine hydrolases [34,38].Membrane fractions were prepared from 293HEK cells transfected with HirFAAH, HirFAAH(S225A), or GFP vector and the relative expression of each protein was determined via western blot (Fig. 5a).These ndings indicated successful transfection and expression for all three proteins.The ActiveX™ TAMRA-FP probe labeled an ~ 83 kDa protein in the HirFAAH expressing membrane fractions that corresponded with the expected molecular weight of GFP tagged HirFAAH (Figs. 5b; N = 9).Hydrolase activity at this molecular weight was not present in any of the GFP lanes (Fig. 5b; N = 7).Next, we tested the effects of URB597, a selective, irreversible inhibitor of mammalian FAAH [39], on the serine hydrolase activity of HirFAAH at concentrations 0.01, 0.1, 1 and 10 µM plus a vehicle control (2% DMSO).A 2-way ANOVA was used to assess serine hydrolase activity in samples from HirFAAH-transfected cells vs. those transfected with the GFP-containing vector and the effects of increasing concentrations of URB97 on hydrolase activity.This analysis detected a signi cant effect of transfection construct with serine hydrolase activity with HirFAAH samples have much greater activity than GFP-only samples (F 1,46 = 16.84,p < 0.0001).Preincubation of sample with URB597 decreased hydrolase activity in the HirFAAH in a concentration-dependent manner that was not observed in the GFP samples (Fig. 5b, c).This was con rmed by a signi cant concentration effect (F 4,46 = 23.16,p < 0.0001) and a signi cant interaction effect (F 4,46 = 22.88, p < 0.0001).Post-hoc analysis indicated signi cant inhibition of HirFAAH activity by 0.1, 1, and 10 µM URB597 (p < 0.0001 for all).No other proteins labeled by the AcivX™ TAMRA-FP probe exhibited any obvious sensitivity to URB597.

AAMCA-based Fluorescence Assay
While the ABPP assay con rms serine hydrolase activity, it does not con rm that HirFAAH actually metabolizes AEA.To address this issue, we used a high-throughput uorescent screening assay developed by Ramarao et.al. that speci cally measures FAAH activity [40].In this assay, FAAH catalyzes the hydrolysis of a non uorescent AAMCA, a substrate that is speci c to FAAH, to produce arachidonic acid and the highly uorescent AMC (excitation 355nm/emission 460nm).
In order to maximize the amount of HirFAAH used in the AAMCA assay and increase the assay's sensitivity, the membrane fraction of HirFAAH was further puri ed into a microsomal fraction.Previous work has shown that FAAH activity is highest in the microsomal fraction [40].As a validation step to assess our microsomal isolation process, the ABPP assays were repeated using microsomal fractions from the transfected 293HEK cells.In these experiments, serine hydrolase activity was compared from microsome samples prepared from cells transfected with HirFAAH (N = 5), the GFP-only vector (N = 2), and HirFAAH(S225A) mutant (N = 4).Consistent with our previous experiments using membrane fraction samples (Fig. 5), the ActiveX™ TAMRA-FP probe labeled a protein (~ 83kDa) in the HirFAAH-expressing microsomal fractions that was not present in the GFP samples (Fig. 6a, b).In addition, no serine hydrolase activity was observed samples transfected with the HirFAAH(S225A) mutant, indicating that the mutation at the proposed active site did disrupt enzyme function.100 nM URB597 signi cantly inhibited HirFAAH activity with no effect on samples from the GFP-only and HirFAAH(S225A) groups.This was con rmed by 2-way ANOVA which showed a signi cant effect of gene product (F 4,46 = 9.68, p < 0.005), URB597 treatment (F 4,46 = 5.36, p < 0.05), and interaction effect (F 4,46 = 7.38, p < 0.01).To summarize, these TAMRA-FP experiments demonstrated that HirFAAH exhibited the expected serine hydrolase activity, and this activity was reduced on a concentration-dependent manner by the FAAH inhibitor URB597.Furthermore, mutation of HirFAAH at the putative active site did prevent serine hydrolase activity.
Next, microsomal fractions from 293HEK cells expressing GFP (N = 3), HirFAAH (N = 4) or HirFAAH(S225A) (N = 4) were incubated with AAMCA plus either 2% DMSO or increasing concentrations of URB597.Figure 6c shows background levels of AMC production in samples expressing GFP alone incubated in 2% DMSO.No statistically signi cant changes in AMC levels was observed in GFP-only sample incubated in any of the URB597 concentrations (1nM, 10nM, 100nM, 1µM and 10µM).Samples containing wildtype HirFAAH and incubated in DMSO showed substantial AMC production over background levels indicating HirFAAH speci c AAMCA hydrolysis (Fig. 6c), consistent with other FAAHs that metabolize AEA.Additionally, there was a statistically signi cant decrease in the AMC production when the HirFAAH microsomal fraction was incubated with URB597 (Fig. 6c).Concentrations ranging from 1 nM to 10 µM URB597 produced signi cant inhibition of enzymatic activity (post hoc p < 0.001).Interestingly, samples in this analysis appeared more sensitive to inhibition by URB597 compared to samples in the ABPP assay.In the AAMCA assay, 1 nM URB597 produced a signi cant decrease in enzymatic activity, whereas a minimum of 100 nM was required to produce a signi cant decrease relative to the vehicle control in the ABPP assays (see Fig. 5c vs. 6c).This may be a consequence of different sensitivities between the AAMCA and ABPP assays.In the case of HirFAAH(S225A) samples incubated in DMSO, AMC production were at background levels (Fig. 6c) indicating that no hydrolysis activity in this mutant FAAH, consistent with the site mutation interfering with enzymatic activity.HirFAAH(S225A)containing samples exhibited no change in AMC production when pre-incubated with increasing concentrations of URB597 (Fig. 6c).Two-way ANOVA showed a signi cant effect of gene product indicating that only HirFAAH-containing samples exhibited enzymatic activity (F 2,48 = 12.47, p < 0.001).
Analysis also showed a signi cant effect of URB597 concentration (F 5,48 = 13.53,p < 0.001) and a signi cant interaction effect (F 10,48 = 13.10,p < 0.001), indicating that the FAAH inhibitor did reduce enzymatic activity of HirFAAH with no effect on the HirFAAH(S225A) mutant or the GFP control.

Effects of FAAH inhibition on synaptic transmission
Next, we wanted to examine whether URB597 has functional effects on the Hirudo CNS consistent with inhibition of FAAH.Therefore, we examined the effects of URB597 on synaptic transmission by pressure sensory neurons (P cells) in acutely isolated Hirudo ganglia.In previous studies, a 15 min AEA application was found to produce long-lasting (1 hour) in P cell synapses [24,41].If URB597 is increasing AEA levels by inhibiting HirFAAH, then one would expect URB597 to mimic the effect of exogenously applied AEA.
EPSP amplitude was recorded prior to and then 1 hour following a 15 min application of 1 µM URB597 (N = 7) since this was a concentration that appeared to effectively inhibit HirFAAH activity (see Figs. 5c  and 6c).Experiments using 1 µM AEA (N = 5) were also conducted in parallel to compare to the URB597 results.The FAAH inhibitor did signi cantly potentiate the P cell EPSP compared to the 0.001% DMSO control group (N = 5) and did so at a level similar to AEA (Fig. 7a, b).One-way ANOVA showed a signi cant effect of treatment group (F 4,25 = 6.55, p < 0.0001), with the AEA-and URB597-treated ganglia exhibiting signi cant potentiation compared to the DMSO control groups (p < 0.05 and < 0.01, respectively).In past experiments, synaptic potentiation by AEA was blocked by the TRPV inhibitor SB366791, indicating that AEA acted on a Hirudo TRPV-like channel [24].Here the ability of 10µM SB366791 to block synaptic potentiation by AEA was con rmed (Fig. 6B; AEA + SB group, N = 6; p < 0.05).
Furthermore, the co-application of the TRPV inhibitor with URB597 also blocked the FAAH inhibitor's capacity to produce synaptic potentiation (URB + SB, N = 7; p < 0.05).
Consistent with prior studies, AEA-induced synaptic potentiation produced no change in paired-pulse facilitation ratio (PPR) compared to the DMSO controls (Fig. 7c).This suggests that synaptic potentiation occurs at the post-synaptic level.As with AEA, URB597 also did not signi cantly change PPR.One-way ANOVA con rmed no signi cant difference in the percent change in PPR across an of the treatment groups (F 4,25 = 1.93, p > 0.05).For all the treatment groups, no change was observed in the input resistance of the postsynaptic cell (Fig. 7d), indicating that the observed increases in EPSP amplitude were not due to changes in the intrinsic excitability of the postsynaptic neurons, at least as measured in the soma (F 4,25 = 1.42, p < 0.05).

Discussion
In this study we identi ed and characterized putative FAAH-encoding gene from the Hirudo transcriptome.The sequence identi ed was ampli ed from the Hirudo CNS and codes for a full-length, functional FAAH protein.Initial sequence alignment of the HirFAAH indicated the greatest similarity to human FAAH-2 isoform.Further comparisons with other FAAH-2 or putative FAAH-2 sequences from a range of vertebrate and invertebrate species supported the conclusion that HirFAAH is a FAAH-2 orthologue.Most invertebrates appear to only possess FAAH-2 isoforms [42] and our database analysis of other invertebrate FAAH's would seem to con rm this.One notable exception is in C. elegans where a recent study actually reported six FAAH isoforms [21], including what appear to be FAAH-1 and FAAH-2 orthologues.It is unclear whether this expansion of FAAH-encoding genes is limited to C. elegans, others in the phylum Nematoda, other ecdysozoan phyla (e.g., Arthropoda), or other invertebrate in general.
Similarly, it is unclear whether the FAAH-1 gene in C. elegans represents a conserved orthologue to vertebrate FAAH-1 or is an example of convergent evolution.Across the vertebrate phyla FAAH-1 and − 2 have a somewhat unusual distribution.Both FAAH-1 and − 2 are present in amphibians, sh, nonplacental mammals, and placental primates.However, FAAH-2 appears to be absent in non-primate placental mammals [37].
Human FAAH-1 and − 2 differ in their catalytic capacity, with FAAH-1 having higher rates of activity [33,35].Another distinction is that while FAAH-1 and − 2 are both membrane bound proteins, they have different orientations with the membrane.FAAH-1 is oriented with its catalytic region facing the cytoplasm while FAAH-2 is oriented facing the luminal compartment of the endoplasmic reticulum [33].FAAH-2 may also be incorporated into lipid droplets within the cytoplasmic compartment [35].In that study, immuno uorescence microscopy showed HsaFAAH2 localized to ring-like structures in the cytoplasm found to be lipid droplets.HirFAAH protein was also found to localized to ring-like structures in the cytoplasm that appeared to be similar lipid droplets.
That HirFAAH is a functional serine hydrolase was con rmed by its activity in the ABPP assay with the ActveX™ TAMRA-FP probe.The TAMRA-FP probe labeled many proteins when incubated with either lysates or membrane fractions from transfected 293HEK cells, however, only one band differed between the GFP-only vector control and HirFAAH expressing cells.Furthermore, the protein differentially labeled by TAMRA-FP corresponded to the predicted molecular weight for the GFP-tagged HirFAAH.The enzymatic activity of HirFAAH was further elucidated using the AAMCA-based uorescence assay that measures the hydrolysis of AAMCA to generate arachidonic acid and the highly uorescent AMC.
Together, these experiments provide two independent assessments of serine hydrolase activity by HirFAAH.As an additional test of HirFAAH biochemical function, the critical catalytic site for serine hydrolase activity was mutated (serine to alanine) to presumably inactivate the protein.In the TAMRA-FP assay of serine hydrolase activity, uorescence at the band corresponding with hirFAAH was reduced to background levels in the HirFAAH(S225A) sample.Similarly, the uorescent signal produced when HirFAAH(S225A) samples is incubated with AAMCA was also signi cantly lowered to background levels.
URB597 is a widely used FAAH inhibitor that is a potent blocker of FAAH-1 and − 2 with IC 50 of approximately 100 nM and 5 nM, respectively [33].Similar concentration dependent inhibition of HirFAAH by URB597 was observed in both the TAMRA-FP and AAMCA-based uorescence assays.Lower concentrations of URB597 were effective in the AAMCA assay compared to the ABPP assy.This may be due to different sensitivities of the two assays or because the AAMCA assay was carried out in microsome fractions that may have more concentrated amounts of FAAH per sample.To our knowledge this is only the second report in which URB597 has been validated to inhibit FAAH in an invertebrate, the other being in the FAAH-2 isoform in C. elegans [21].This represents an important validation for using URB597 in future studies in Hirudo focusing on the neurobehavioral effects of AEA.
Given that URB597-blocking of FAAH presumably increases AEA levels, we examined whether this drug mimicked the synaptic effects of exogenously applied AEA.In P cell synapses, AEA elicits persistent potentiation and similar increases in the P-to-AP EPSP were observed following URB597 treatment.Furthermore, the URB597-induced potentiation was blocked by co-treatment with the TRPV inhibitor SB366791, again consistent with the effects of AEA which are also appears to be mediated by Hirudo TRPV-like receptor [24,41].It is well-established that TRPV1 acts as a cannabinoid receptor in mammals [43,44] and our studies to date suggest that a TRPV like channel mediates the effects of both 2-AG and AEA in Hirudo [23,41,45].Neither AEA nor URB597 elicited a change in PPR, indicating the site of potentiation was postsynaptic.This agrees with our previous studies of AEA modulation of the P synapses which lacks TRPV-like channels.Potentiation of P synapses is mediated rst by a depression of tonic inhibition of the P cell by AEA [46].The resulting cannabinoid-mediated disinhibition "gates" synaptic potentiation via a mechanism that is dependent on NMDA receptor activation and CamKII [47].
Endocannabinoid signaling in general, and AEA in particular, play an important role in a variety of behavioral and physiological processes across the animal kingdom.FAAH plays an important role in regulating AEA levels and represents a "druggable" site for potentially raising AEA levels to produce a clinical effect, e.g., treating chronic pain.In preclinical studies, increasing AEA levels via pharmacological inhibitors or genetic knockdown of FAAH has been shown to produce analgesic effects [48,49].This approach is further supported by a single clinical case of chronic insensitivity to pain that involves a mutation reducing FAAH activity and raising AEA levels [50].However, an attempt to develop an effective FAAH inhibitor (PF-04457845) as a treatment for chronic pain was unsuccessful due to lack of e cacy [51].There are undoubtably many reasons for this disconnect between preclinical studies and clinical results.However, one element that we have focused on is that endocannabinoids can have both pro-and anti-nociceptive effects, a nding supported by our work in Hirudo and a number of other ndings in mammals [12,52,53].In Hirudo we have found the both 2-AG and AEA depress nociceptive synapses, producing an anti-nociceptive effect, but disinhibit/potentiate non-nociceptive synapses, producing a pronociceptive effect [24,27,46,47].Similar observations have also been observed in the mammalian spinal circuitry [54,55].In Hirudo, different patterns of afferent activity elicit either the pro-or anti-nociceptive effects of endocannabinoids [24,25,27,56].An approach we plan to investigate in the future is whether pairing FAAH inhibition, e.g., URB597, with certain patterns of activity that elicit endocannabinoid synthesis, e.g., repetitive non-nociceptive afferent stimulation, can selectively produce anti-nociceptive effects.The ndings from these studies may inform how best to deploy FAAH inhibitors in the clinical setting as a treatment for chronic pain.

Declarations
Statements and Declarations: This work was supported by grants from to BDB from the NINDS (1R01NS092716-01A1) and to RTF from NSF-DGE (DGE-1545679).
Competing Interests: The authors have no nancial interests to disclose.
Author Contributions: All authors contributed to the study conception and design.Material preparation, data collection and analysis were performed by Emily Kabeiseman, Riley Paulson and Brian Burrell.The rst draft of the manuscript was written by Emily Kabeiseman and Brian Burrell.       Figure 7

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Figure 1 Sequence
Figure 1

Table 1
5 g/L Instant Ocean Sea Salt, Aquarium Systems) on a 12-hour light/dark cycle in a 15°C incubator.Animals were anesthetized in 15mM MgCl 2 saline with 5% ethanol at 4°C for thirty minutes.Individual ganglia were dissected and pinned into 35mm Sylgard-lined dishes for electrophysiology experiments under constant perfusion Hirudo saline solution (110mM NaCl, 4mM KCl, 1.8mM CaCl 2 , 1mM MgCl 2 ,