Recently, covalent drugs have attracted great interest in the drug discovery community, with successful examples that have demonstrated their therapeutic effects. Here, we focus on the covalent inhibition of the fatty acid amide hydrolase (FAAH), which is a promising strategy in the treatment of pain and inflammation. Among the most recent and potent FAAH inhibitors (FAAHi), there are the cyclic piperidine/piperazine aryl ureas. FAAH hydrolyzes efficiently the amide bond of these compounds, forming a covalent enzyme-inhibitor adduct. To rationalize this experimental evidence, we performed an extensive computational analysis centered on the piperidine-based PF750 (1) and the piperazine-based JNJ1661010 (2), two potent lead compounds used to generating covalent inhibitors as clinical candidates. We found that FAAH induces a distortion of the amide bond of the piperidine/piperazine aryl ureas. QM/MM ΔELUMO-HOMO energies indicate that the observed enzyme-induced distortion of the amide bond favors the formation of a covalent FAAH- inhibitor adduct. These findings could help in the rational structure-based design of novel covalent FAAHi.
The “thrifty gene hypothesis” posits that evolution preferentially selects physiological mechanisms that optimize energy storage to increase survival under alternating conditions of abundance and scarcity of food. Recent experiments suggest that endocannabinoids – a class of lipid-derived mediators that activate cannabinoid receptors in many cells of the body – are key agents of energy conservation. The new evidence indicates that these compounds increase energy intake and decrease energy expenditure by controlling the activity of peripheral and central neural pathways involved in the sensing and hedonic processing of sweet and fatty foods, as well as in the storage of their energy content for future use.
2-arachidonoylglycerol; cannabinoid receptors; dietary fat
Alkylcarbamic acid biphenyl-3-yl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors that comprises cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester (URB597), a compound with analgesic, anxiolytic-like and antidepressant-like properties in rat and mouse models. Here, we extended the structure-activity relationships (SARs) for this class of compounds by replacing the cyclohexyl ring of the parent compound cyclohexylcarbamic acid biphenyl-3-yl ester (URB524) (IC50, for FAAH = 63 nM) with a selected set of substituents of different size, shape, flexibility and lipophilicity.
Docking experiments and Linear Interaction Energy (LIE) calculations indicated that the N-terminal group of O-arylcarbamates fits within the lipophilic region of the substrate-binding site, mimicking the arachidonoyl chain of anandamide. Significant potency improvements were observed for the β-naphthylmethyl derivative 4q (IC50 = 5.3 nM) and its 3′-carbamoylbiphenyl-3-yl ester 4z (URB880, IC50 = 0.63 nM), indicating that shape complementarity and hydrogen bonds are crucial to obtain highly potent inhibitors.
Fatty-acid amide hydrolase (FAAH) catalyzes the intracellular hydrolysis of the endocannabinoid anandamide and other bioactive lipid amides. In the present study, we conducted a comparative characterization of the effects of the newly identified brain-impermeant FAAH inhibitor, URB937 ([3-(3-carbamoylphenyl)-4-hydroxy-phenyl] N-cyclohexylcarbamate), in various rodent models of acute and persistent pain. When administered by the oral route in mice, URB937 was highly active (median effective dose, ED50, to inhibit liver FAAH activity: 0.3 mg-kg−1) and had a bioavailability of 5.3%. The antinociceptive effects of oral URB937 were investigated in mouse models of acute inflammation (carrageenan), peripheral nerve injury (chronic sciatic nerve ligation) and arthritis (complete Freund’s adjuvant). In all models, URB937 was as effective or more effective than standard analgesic and anti-inflammatory drugs (indomethacin, gabapentin, dexamethasone) and reversed pain-related responses (mechanical hyperalgesia, thermal hyperalgesia, and mechanical allodynia) in a dose-dependent manner. ED50 values ranged from 0.2 to 10 mg-kg−1, depending on model and readout. Importantly, URB937 was significantly more effective than two global FAAH inhibitors, URB597 and PF-04457845, in the complete Freund’s adjuvant model. The effects of a combination of URB937 with the non-steroidal anti-inflammatory agent, indomethacin, were examined in the carrageenan and chronic sciatic nerve ligation models. Isobolographic analyses showed that the two compounds interacted synergistically to attenuate pain-related behaviors. Furthermore, URB937 reduced the number and severity of gastric lesions produced by indomethacin, while exerting no ulcerogenic effect when administered alone. The results indicate that the peripheral FAAH inhibitor URB937 is more effective than globally active FAAH inhibitors at inhibiting inflammatory pain. Our findings further suggest that FAAH and cyclooxygenase inhibitors interact functionally in peripheral tissues, to either enhance or hinder each other’s actions.
inflammation; anandamide; neuropathic pain; gastric lesions; cannabinoid receptors
The endocannabinoids and their attending CB1 cannabinoid receptors have been implicated in the control of cognition, but their possible roles in dementias are still unclear. In the present study, we used liquid chromatography/mass spectrometry to conduct an endocannabinoid-targeted lipidomic analysis of post mortem brain samples from 38 Alzheimer’s disease (AD) patients and 17 control subjects, matched for age and post mortem interval. The analysis revealed that midfrontal and temporal cortex tissue from AD patients contains, relative to control subjects, significantly lower levels of the endocannabinoid anandamide and its precursor 1-stearoyl, 2-docosahexaenoyl-sn-glycero-phosphoethanolamine-N-arachidonoyl (NArPE). No such difference was observed with the endocannabinoid 2-arachidonoyl-sn-glycerol or 15 additional lipid species. In AD patients, but not in control subjects, statistically detectable positive correlations were found between (a) anandamide content in midfrontal cortex and scores of the Kendrick’s digit copying test (P=0.004, r=0.81; n=10), which measures speed of information processing; and (b) anandamide content in temporal cortex and scores of the Boston naming test (P=0.027, r=0.52; n=18), which assesses language facility. Furthermore, anandamide and NArPE levels in midfrontal cortex of the study subjects inversely correlated with levels of the neurotoxic amyloid peptide, Aβ42, while showing no association with Aβ40 levels, amyloid plaque load or tau protein phosphorylation. Finally, high endogenous levels of Aβ42 in APPSWE/Neuro-2a cells directly reduced anandamide and NArPE concentrations in cells lysates. The results suggest that an Aβ42-dependent impairment in brain anandamide mobilization contributes to cognitive dysfunction in AD.
endocannabinoid; anandamide; amyloid β42; cognitive dysfunction; Alzheimer’s disease; human brain; lipidomics
The endocannabinoid system plays a critical role in the control of energy homeostasis, but the identity and localization of the endocannabinoid signal involved remain unknown. In the present study we developed transgenic mice that over-express in forebrain neurons the presynaptic hydrolase, monoacylglycerol lipase (MGL), which deactivates the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). MGL-overexpressing mice show a 50 percent decrease in forebrain 2-AG levels, but no overt compensation in other endocannabinoid components. This biochemical abnormality is accompanied by a series of metabolic changes that include leanness, elevated energy cost of activity and hypersensitivity to β3-adrenergic-stimulated thermogenesis, which is corrected by reinstating 2-AG activity at CB1-cannabinoid receptors. Additionally, the mutant mice are resistant to diet-induced obesity and express high levels of thermogenic proteins, such as uncoupling protein-1, in their brown adipose tissue. The results suggest that 2-AG signaling through CB1 regulates the activity of forebrain neural circuits involved in the control of energy dissipation.
The O-arylcarbamate URB937 is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the deactivation of the endocannabinoid anandamide. URB937 is unique among FAAH inhibitors in that is actively extruded from the central nervous system (CNS), and therefore increases anandamide levels exclusively in peripheral tissues. Despite its limited distribution, URB937 exhibits marked analgesic properties in rodent models of pain. Pharmacological evidence suggests that the extrusion of URB937 from the CNS may be mediated by the ABC membrane transporter ABCG2 (also called Breast Cancer Resistance Protein, BCRP). In the present study, we show that URB937 is a substrate for both mouse and human orthologues of ABCG2. The relative transport ratios for URB937 in Madin-Darby canine kidney (MDCKII) cells monolayers over-expressing either mouse Abcg2 or human ABCG2 were significantly higher compared to parental monolayers (13.6 and 13.1 vs 1.5, respectively). Accumulation of the compound in the luminal/apical side was prevented by co-administration of the selective ABCG2 inhibitor, Ko-143. In vivo studies in mice showed that URB937 (25 mg-kg−1) readily entered the brain and spinal cord of Abcg2-deficient mice following intraperitoneal administration, whereas the same dose of drug remained restricted to peripheral tissues in wild-type mice. By identifying ABCG2 as a transport mechanism responsible for the extrusion of URB937 from the CNS, the present results should facilitate the rational design of novel peripherally restricted FAAH inhibitors.
Fatty-acid amide hydrolase; URB937; Breast cancer resistance protein; blood-brain barrier; central nervous system; Abcg2-deficient mice
Secondary alkylcarbamic acid biphenyl-3-yl esters are a class of Fatty Acid Amide Hydrolase (FAAH) inhibitors, which include the reference compounds URB597 and URB694. Given the intrinsic reactivity of the carbamate group, the in vivo potency of these molecules in rats is strongly affected by their hydrolysis in plasma or hepatic metabolism. In the present study, in vitro chemical and metabolic stability assays (rat plasma and rat liver S9 fraction) were used to investigate the structure-property relationships (SPRs) for a focused series of title compounds, where lipophilicity and steric hindrance of the carbamate N-substituent had been modulated. The resulting degradation rates indicate that a secondary or tertiary alkyl group at the carbamate nitrogen atom increases hydrolytic stability towards rat plasma esterases. The calculated solvent accessible surface area (SASA) of the carbamate fragment was employed to describe the differences observed in rate constants of hydrolysis in rat plasma (log kplasma), suggesting that stability in plasma increases if the substituent exerts a shielding effect on the carbamate carbonyl. Stability in rat liver S9 fraction is increased when a tertiary carbon is bound to the carbamate nitrogen atom, while other steric effects showed complex relationships with degradation rates. The SPRs here described may be applied at the pharmacokinetic optimization of other classes of carbamate FAAH inhibitors.
alkylcarbamates; FAAH inhibitors; liquid chromatography; SPR; stability; rat plasma
FAAH is the main degrading enzyme of the fatty acid ethanolamides anandamide (AEA) and oleoylethanolamide (OEA), which have opposite effects on food intake and energy balance. AEA, an endogenous ligand of CB1 cannabinoid receptors, enhances food intake and energy storage, whereas OEA binds to PPAR-α receptors to reduce food intake and promoting lipolysis. To elucidate the role of FAAH in food intake and energy balance, we have evaluated different metabolic and behavioral responses related to feeding in FAAH-deficient (FAAH−/−) mice and their wild-type littermates. Total daily food intake was similar in both genotypes, but high-fat food consumption was enhanced during the dark hours and decreased during the light hours in FAAH−/− mice. The reinforcing and motivational effects of food were also enhanced in FAAH−/− mice as revealed by operant behavioral paradigms. These behavioral responses were reversed by the administration of the selective CB1 cannabinoid antagonist rimonabant. Furthermore, body weight, total amount of adipose tissue, plasmatic free fatty acids and triglyceride content in plasma, liver, skeletal muscle and adipose tissue, were increased in FAAH−/− mice. Accordingly, leptin levels were increased and adiponectin levels decreased in these mutant FAAH−/− mice also showed enhanced plasmatic insulin and blood glucose levels revealing an insulin resistance. As expected, both AEA and OEA levels were increased in hypothalamus, small intestine and liver of FAAH−/− mice. These results indicate that the lack of FAAH predominantly promotes energy storage by food intake-independent mechanisms, through the enhancement of AEA levels rather than promoting the anorexic effects of OEA.
fatty acid amide hydrolase; anandamide; oleoylethanolamide; food intake; body weight; lipid turnover; food reinforcement
We discuss and present new data regarding the physiological and molecular mechanisms of nuclear receptor activation in pain control, with a particular emphasis on non-genomic effects of ligands at peroxisome proliferator-activated receptor (PPAR), GPR30, and classical estrogen receptors. PPARα agonists rapidly reduce both acute and chronic pain in a number of pain assays. These effects precede transcriptional anti-inflammatory actions, and are mediated in part by IKca and BKca channels on DRG neurons. In contrast to the peripheral site of action of PPARα ligands, the dorsal horn supports the expression of PPARγ. Intrathecal administration of PPARγ ligands rapidly (≤5 minutes) attenuated mechanical and thermal hypersensitivity associated with nerve injury in a dose-dependent manner that could be blocked with PPARγ antagonists. By contrast, a PPARγ antagonist itself rapidly increased the mechanical allodynia associated with nerve injury. These data suggest that ligand-dependent, non-genomic activation of spinal PPARγ decreases behavioral signs of inflammatory and neuropathic pain. We also report that the GPR30 is expressed on cultured sensory neurons, that activation of the receptor elicits signaling to increase calcium accumulation and PKCε translocation, and that this signaling may contribute to increased neuronal sensitivity as treatment with the GPR30 agonist induces hyperalgesia. Finally, application of the membrane-impermeable 17ß-E2-BSA rapidly (within 15 min) enhanced BK-stimulated inositol phosphate (IP) accumulation and PGE2-mediated cAMP accumulation in trigeminal ganglion cultures. We conclude that nuclear receptor ligands may operate through rapid, non-genomic mechanisms to modulate inflammatory and neuropathic pain.
Cannabinoid CB1 receptor antagonists reduce body weight in rodents and humans, but their clinical utility as anti-obesity agents is limited by centrally mediated side effects. Here, we describe the first mixed CB1 antagonist/CB2 agonist, URB447 ([4-amino-1-(4-chlorobenzyl)-2-methyl-5-phenyl-1H-pyrrol-3-yl](phenyl)methanone), which lowers food intake and body-weight gain in mice without entering the brain or antagonizing central CB1-dependent responses. URB447 may provide a useful pharmacological tool for investigating the cannabinoid system, and might serve as a starting point for developing clinically viable CB1 antagonists devoid of central side effects.
URB447; Peripheral cannabinoid antagonism; Anti-obesity agents; Pyrrolic CB-antagonists
Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in controlling pain sensitivity in vivo. Here we show that foot shock stress produces antinociception in rats by activating type-5 metabotropic glutamate receptors (mGlu5) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing 2-AG. Stimulation of mGlu5 in the dlPAG with (S)-3,5-dihydroxyphenylglycine (DHPG) triggered 2-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases 2-AG, and presynaptic CB1 cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80267 and (−)-tetrahydrolipstatin (THL), which inhibit activity of DGL-α and DGL-β isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of 2-AG without altering levels of anandamide. The putative 2-AG-synthesizing enzyme DGL-α co-localized with mGlu5 at postsynaptic sites of the dlPAG, whereas CB1 was confined to presynaptic terminals, consistent with a role for 2-AG as a retrograde signaling messenger. Finally, virally-mediated silencing of DGL-α, but not DGL-β, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and 2-AG formation. The results indicate that activation of the postsynaptic mGlu5–DGL-α cascade triggers retrograde 2-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.
2-arachidonoylglycerol; metabotropic glutamate receptor; retrograde signaling; endocannabinoid; pain; antinociception
The amplitude of the depolarization-evoked Ca2+ transient is larger in dorsal root ganglion (DRG) neurons from tumor-bearing mice compared to that of neurons from naive mice, and the change is mimicked by co-culturing DRG neurons with the fibrosarcoma cells used to generate the tumors (Khasabova et al., 2007). The effect of palmitoylethanolamide (PEA), a ligand for the peroxisome proliferator-activated receptor-alpha (PPARα), was determined on the evoked-Ca2+ transient in the co-culture condition. The level of PEA was reduced in DRG cells from tumor-bearing mice as well as those co-cultured with fibrosarcoma cells. Pretreatment with PEA, a synthetic PPARα agonist (GW7647), or ARN077, an inhibitor of the enzyme that hydrolyses PEA, acutely decreased the amplitude of the evoked Ca2+ transient in small DRG neurons co-cultured with fibrosarcoma cells. The PPARα antagonist GW6471 blocked the effect of each. In contrast, the PPARα agonist was without effect in the control condition, but the antagonist increased the amplitude of the Ca2+ transient suggesting that PPARα receptors are saturated by endogenous ligand under basal conditions. Effects of drugs on mechanical sensitivity in vivo paralleled their effects on DRG neurons in vitro. Local injection of ARN077 decreased mechanical hyperalgesia in tumor-bearing mice, and the effect was blocked by GW6471. These data support the conclusion that the activity of DRG neurons is rapidly modulated by PEA through a PPARα-dependent mechanism. Moreover, agents that increase the activity of PPARα may provide a therapeutic strategy to reduce tumor-evoked pain.
The combination of memantine, an N-methyl-d-aspartate (NMDA) receptor antagonist, with an acetylcholinesterase inhibitor (AChEI) is the current standard of care in Alzheimer's disease (AD). Galantamine, an AChEI currently marketed for the treatment of AD, exerts memory-enhancing and neuroprotective effects via activation of nicotinic acetylcholine receptors (nAChRs). Here, we investigated the neuroprotective properties of galantamine in primary cultures of rat cortical neurons when given alone or in combination with memantine. In agreement with previous findings, we found that memantine was fully effective in reversing NMDA toxicity at concentrations of 2.5 and 5 μmol/L. Galantamine also completely reversed NMDA toxicity at a concentration of 5 μmol/L. The α7 and α4β2 nAChR antagonists, methyllycaconitine, and dihydro-β-erythroidine blocked the neuroprotective effect of galantamine, demonstrating the involvement of nAChRs. The combination of memantine with galantamine produced synergistic actions, such that full neuroprotective efficacy, was obtained at inactive concentrations of memantine (0.1 μmol/L) and galantamine (1 μmol/L). A similar potentiation was also observed when memantine was replaced with ifenprodil, suggesting a possible involvement of the NR2B subunit of the NMDA receptor. In summary, our study reports for the first time at a cellular level that memantine and galantamine interact on the same excitotoxic cascade and that the combination of these two drugs can result in a remarkable neuroprotective effect.
Alzheimer's disease; drug combination; NMDA neurotoxicity; NR2B; polypharmacology; primary cortical neurons
Alzheimer's disease (AD) is characterized by progressive loss of cognitive function, dementia and altered behavior. Over 30 million people worldwide suffer from AD and available therapies are still palliative rather than curative. Recently, Memoquin (MQ), a quinone-bearing polyamine compound, has emerged as a promising anti-AD lead candidate, mainly thanks to its multi-target profile. MQ acts as an acetylcholinesterase and β-secretase-1 inhibitor, and also possesses anti-amyloid and anti-oxidant properties. Despite this potential interest, in vivo behavioral studies with MQ have been limited. Here, we report on in vivo studies with MQ (acute and sub-chronic treatments; 7–15 mg/kg per os) carried out using two different mouse models: i) scopolamine- and ii) beta-amyloid peptide- (Aβ-) induced amnesia. Several aspects related to memory were examined using the T-maze, the Morris water maze, the novel object recognition, and the passive avoidance tasks. At the dose of 15 mg/kg, MQ was able to rescue all tested aspects of cognitive impairment including spatial, episodic, aversive, short and long-term memory in both scopolamine- and Aβ-induced amnesia models. Furthermore, when tested in primary cortical neurons, MQ was able to fully prevent the Aβ-induced neurotoxicity mediated by oxidative stress. The results support the effectiveness of MQ as a cognitive enhancer, and highlight the value of a multi-target strategy to address the complex nature of cognitive dysfunction in AD.
Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in controlling pain sensitivity in vivo. Here, we show that footshock stress produces antinociception in rats by activating type 5 metabotropic glutamate receptors (mGlu5) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing 2-AG. Stimulation of mGlu5 in the dlPAG with DHPG [(S)-3,5-dihydroxyphenylglycine] triggered 2-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases 2-AG, and presynaptic CB1 cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80267 [1,6-bis(cyclohexyloximinocarbonylamino)hexane] and (−)-tetrahydrolipstatin (THL), which inhibit activity of DGL-α and DGL-β isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of 2-AG without altering levels of anandamide. The putative 2-AG-synthesizing enzyme DGL-α colocalized with mGlu5 at postsynaptic sites of the dlPAG, whereas CB1 was confined to presynaptic terminals, consistent with a role for 2-AG as a retrograde signaling messenger. Finally, virally mediated silencing of DGL-α, but not DGL-β, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and 2-AG formation. The results indicate that activation of the postsynaptic mGlu5–DGL-α cascade triggers retrograde 2-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.
The expression of acid ceramidase (AC) – a cysteine amidase that hydrolyses the proapoptotic lipid ceramide – is abnormally high in several human tumors, which is suggestive of a role in chemoresistance. Available AC inhibitors lack, however, the potency and drug-likeness necessary to test this idea. Here we show that the antineoplastic drug carmofur, which is used in the clinic to treat colorectal cancers, is a potent AC inhibitor and that this property is essential to its anti-proliferative effects. Modifications in the chemical scaffold of carmofur yield new AC inhibitors that act synergistically with standard antitumoral drugs to prevent cancer cell proliferation. These findings identify AC as an unexpected target for carmofur, and suggest that this molecule can be used as starting point for the design of novel chemosensitizing agents.
Cyclohexylcarbamic acid aryl esters are a class of Fatty Acid Amide Hydrolase (FAAH) inhibitors, which includes the reference compound URB597. The reactivity of their carbamate fragment is involved in pharmacological activity and may affect pharmacokinetic and toxicological properties. We conducted in vitro stability experiments in chemical and biological environments to investigate the structure-stability relationships in this class of compounds. The results show that electrophilicity of the carbamate influences its chemical stability, as suggested by the relation between the rate constant of alkaline hydrolysis (log kpH9) and the energy of lowest unoccupied molecular orbital (LUMO). Introduction of small, electron donor substituents at conjugated positions of the O-aryl moiety increased overall hydrolytic stability of the carbamate group without affecting FAAH inhibitory potency, whereas peripheral nonconjugated hydrophilic groups, which favor FAAH recognition, helped reducing oxidative metabolism in the liver.
FAAH inhibitors; cyclohexylcarbamic acid aryl esters; structure-activity relationships; stability; liquid chromatography
Preclinical and clinical evidence suggests that docosahexaenoic acid (DHA), an omega-3 fatty acid derived from diet or synthesized in the liver, decreases the risk of developing Alzheimer's disease (AD). DHA levels are reduced in the brain of subjects with AD, but it is still unclear whether human dementias are associated with dysregulations of DHA metabolism. A systems biological view of omega-3 fatty acid metabolism offered unexpected insights on the regulation of DHA homeostasis in AD1. Results of multi-organ lipidomic analyses were integrated with clinical and gene-expression data sets to develop testable hypotheses on the functional significance of lipid abnormalities observed and on their possible mechanistic bases. One surprising outcome of this integrative approach was the discovery that the liver of AD patients has a limited capacity to convert shorter chain omega-3 fatty acids into DHA due to a deficit in the peroxisomal D-bifunctional protein. This deficit may contribute to the decrease in brain DHA levels and contribute to cognitive impairment.
Despite several recent studies suggesting that dysregulation of brain lipid metabolism might contribute to the mechanisms of aging and Alzheimer’s disease (AD), lipid metabolism has not been evaluated extensively in the aging brain. Here, we use a lipidomic approach to demonstrate that antioxidants plus mitochondrial cofactors treatment, either alone or in combination with behavioral enrichment, attenuates lipid abnormalities in the frontal cortices of aged canine in a manner correlated with cognitive scores. Our analyses revealed that the levels of free palmitoleic acid and nervonic acid were decreased in frontal cortices of aged dogs (n=5-6/group) treated with antioxidant compared to the control group. The monounsaturated/saturated fatty acid ratio, also known as ‘desaturation index’ - an ex-vivo indicator of stearoyl-CoA desaturase activity, was also reduced in the frontal cortex of dogs treated with antioxidants compared to control groups. Increased palmitoleic acid levels and desaturation index were positively correlated with increased reversal learning errors and decreased cognitive performance. In conclusion, our study indicates that the addition of antioxidants and mitochondrial cofactors to the regular diet alters the composition of free fatty acids in the aged brain. Together with data showing increased palmitoleic acid levels in AD patients, our data suggest that reducing palmitoleic acid levels and desaturation index in the brain may be associated with improved cognitive performance.
Lipids; canine; aging; Alzheimer’s disease; SCD; frontal cortex
Rodent experiments have emphasized a role of central fatty acid (FA) species, such as oleic acid, in regulating peripheral glucose and energy metabolism. Thus, we hypothesized that central FAs are related to peripheral glucose regulation and energy expenditure in humans. To test this we measured FA species profiles in cerebrospinal fluid (CSF) and plasma of 32 individuals who stayed in our clinical inpatient unit for 6 days. Body composition was measured by dual energy X-ray absorptiometry and glucose regulation by an oral glucose test (OGTT) followed by measurements of 24 hour (24EE) and sleep energy expenditure (SLEEP) as well as respiratory quotient (RQ) in a respiratory chamber. CSF was obtained via lumbar punctures; FA concentrations were measured by liquid chromatography/mass spectrometry. As expected, FA concentrations were higher in plasma compared to CSF. Individuals with high concentrations of CSF very-long-chain saturated FAs had lower rates of SLEEP. In the plasma moderate associations of these FAs with higher 24EE were observed. Moreover, CSF monounsaturated long-chain FA (palmitoleic and oleic acid) concentrations were associated with lower RQs and lower glucose area under the curve during the OGTT. Thus, FAs in the CSF strongly correlated with peripheral metabolic traits. These physiological parameters were most specific to long-chain monounsaturated (C16∶1, C18∶1) and very-long-chain saturated (C24∶0, C26∶0) FAs. Conclusions: Together with previous animal experiments these initial cross-sectional human data indicate that central FA species are linked to peripheral glucose and energy homeostasis.
To celebrate the first 10 years of Nature Reviews Neuroscience, we invited the authors of the most cited article of each year to look back on the state of their field of research at the time of publication and the impact their article has had, and to discuss the questions that might be answered in the next 10 years. This selection of highly cited articles provides interesting snapshots of the progress that has been made in diverse areas of neuroscience. They show the enormous influence of neuroimaging techniques and highlight concepts that have generated substantial interest in the past decade, such as neuroimmunology, social neuroscience and the `network approach' to brain function. These advancements will pave the way for further exciting discoveries that lie ahead.
The endocannabinoid anandamide is removed from the synaptic space by a selective transport system, expressed in neurons and astrocytes, which remains molecularly uncharacterized. Here we describe a partly cytosolic variant of the intracellular anandamide-degrading enzyme, fatty acid amide hydrolase-1 (FAAH-1), termed FAAH-like anandamide transporter (FLAT), which lacks amidase activity but binds anandamide with low micromolar affinity and facilitates its translocation into cells. Known anandamide transport inhibitors, such as AM404 and OMDM-1, block these effects. Additionally, we identify a competitive antagonist of the interaction of anandamide with FLAT, the phthalazine derivative ARN272, which prevents anandamide internalization in vitro, interrupts anandamide deactivation in vivo, and exerts profound analgesic effects in rodent models of nociceptive and inflammatory pain, which are mediated by CB1 cannabinoid receptors. The results identify FLAT as a critical molecular component of anandamide transport in neural cells and a potential target for therapeutic drugs.
Recent findings indicate that inhibitors of fatty acid amide hydrolase (FAAH) counteract the rewarding effects of nicotine in rats. FAAH inhibition increases levels of several endogenous substances in the brain, including the endocannabinoid anandamide and the non-cannabinoid fatty-acid ethanolamides oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), which are ligands for alpha-type peroxisome proliferator-activated nuclear receptors (PPAR-α). Here, we evaluated whether directly-acting PPAR-α agonists can modulate reward-related effects of nicotine.
We combined behavioral, neurochemical and electrophysiological approaches to evaluate effects of the PPAR-α agonists WY14643 and methOEA (a long-lasting form of OEA) on: (1) nicotine self-administration in rats and squirrel monkeys; (2) reinstatement of nicotine-seeking behavior in rats and monkeys; (3) nicotine discrimination in rats; (4) nicotine-induced electrophysiological activity of VTA dopamine neurons in anesthetized rats; and (5) nicotine-induced elevation of dopamine levels in the nucleus accumbens shell of freely-moving rats.
PPAR-α agonists dose-dependently decreased nicotine self-administration and nicotine-induced reinstatement in rats and monkeys, but did not alter food- or cocaine-reinforced operant behavior or the interoceptive effects of nicotine. PPAR-α agonists also dose-dependently decreased nicotine-induced excitation of dopamine neurons in the ventral tegmental area (VTA) and nicotine-induced elevations of dopamine levels in the nucleus accumbens shell of rats. The ability of WY14643 and methOEA to counteract the behavioral, electrophysiological, and neurochemical effects of nicotine was reversed by the PPAR-α antagonist MK886.
These findings indicate that PPAR-α might provide a valuable new target for anti-smoking medications.
Nicotine; PPAR-α; reinstatement; reward; OEA; PEA; FAAH
The N-terminal nucleophile (Ntn) hydrolases are a superfamily of enzymes specialized in the hydrolytic cleavage of amide bonds. Even though several members of this family are emerging as innovative drug targets for cancer, inflammation, and pain, the processes through which they catalyze amide hydrolysis remains poorly understood. In particular, the catalytic reactions of cysteine Ntn-hydrolases have never been investigated from a mechanistic point of view. In the present study, we used free energy simulations in the quantum mechanics/molecular mechanics framework to determine the reaction mechanism of amide hydrolysis catalyzed by the prototypical cysteine Ntn-hydrolase, conjugated bile acid hydrolase (CBAH). The computational analyses, which were confirmed in water and using different CBAH mutants, revealed the existence of a chair-like transition state, which might be one of the specific features of the catalytic cycle of Ntn-hydrolases. Our results offer new insights on Ntn-mediated hydrolysis and suggest possible strategies for the creation of therapeutically useful inhibitors.