DO34

Inhibition of Diacylglycerol Lipase Impairs Fear Extinction in Mice

Elucidating the underlying molecular mechanisms regulating fear and extinction learning may offer insights that can lead to novel treatments for debilitating anxiety and trauma-related disorders including posttraumatic stress disorder. The endocannabinoid (eCB) system is a retrograde inhibitory signaling pathway involved in regulating central responses to stress. The eCB 2-arachidonoylglycerol (2-AG) has recently been proposed to serve as a homeostatic signal mitigating adverse effects of stress exposure, however, less well understood is 2-AG's role in fear learning and fear extinction. In this study, we have sought to explore 2-AG's role in fear conditioning and fear extinction by disrupting 2-AG synthesis utilizing the DAGL inhibitor (DO34) and DAGLα knock-out mice (DAGLα-/-). We found that DAGLα-/- mice, and male and female C57B6/J mice treated with DO34, exhibited impairment in extinction learning in an auditory cue fear-conditioning paradigm. DO34 did not increase unconditioned freezing. Interestingly, inhibition of fatty-acid amide hydrolase was not able to restore normal fear extinction in DO34-treated mice suggesting increased Anandamide cannot compensate for deficient 2-AG signaling in the regulation of fear extinction. Moreover, augmentation of CB1R signaling with tetrahydrocannabinol also failed to restore normal fear extinction in DO34-treated mice. Overall, these data support the hypothesis that DAGLα plays an important role in fear extinction learning. Although genetic and pharmacological disruption of DAGL activity causes widespread lipidomic remodeling, these data combined with previous studies putatively suggest that deficient 2-AG signaling could be a susceptibility endophenotype for the development of trauma-related psychiatric disorders.

Investigation of Diacylglycerol Lipase Alpha Inhibition in the Mouse Lipopolysaccharide Inflammatory Pain Model

Diacylglycerol lipase (DAGL) α and β, the major biosynthetic enzymes of the endogenous cannabinoid (endocannabinoid) 2-arachidonylglycerol (2-AG), are highly expressed in the nervous system and immune system, respectively. Genetic deletion or pharmacological inhibition of DAGL-β protects against lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal macrophages and reverses LPS-induced allodynia in mice. To gain insight into the contribution of DAGL-α in LPS-induced allodynia, we tested global knockout mice as well as DO34, a dual DAGL-α/β inhibitor. Intraperitoneal administration of DO34 (30 mg/kg) significantly decreased whole-brain levels of 2-AG (?83%), anandamide (?42%), and arachidonic acid (?58%). DO34 dose-dependently reversed mechanical and cold allodynia, and these antinociceptive effects did not undergo tolerance after 6 days of repeated administration. In contrast, DO34 lacked acute thermal antinociceptive, motor, and hypothermal pharmacological effects in naive mice. As previously reported, DAGL-β (-/-) mice displayed a protective phenotype from LPS-induced allodynia. However, DAGL-α (-/-) mice showed full allodynic responses, similar to their wild-type littermates. Interestingly, DO34 (30 mg/kg) fully reversed LPS-induced allodynia in DAGL-α (+/+) and (-/-) mice, but did not affect the antinociceptive phenotype of DAGL-β (-/-) mice in this model, indicating a DAGL-α-independent site of action. These findings suggest that DAGL-α and DAGL-β play distinct roles in LPS-induced nociception. Whereas DAGL-α appears to be dispensable for the development and expression of LPS-induced nociception, DAGL-β inhibition represents a promising strategy to treat inflammatory pain.

Diacylglycerol Lipase-Alpha Regulates Hippocampal-Dependent Learning and Memory Processes in Mice

Diacylglycerol lipase-α (DAGL-α), the principal biosynthetic enzyme of the endogenous cannabinoid 2-arachidonylglycerol (2-AG) on neurons, plays a key role in CB₁ receptor-mediated synaptic plasticity and hippocampal neurogenesis, but its contribution to global hippocampal-mediated processes remains unknown. Thus, the present study examines the role that DAGL-α plays on LTP in hippocampus, as well as in hippocampal-dependent spatial learning and memory tasks, and on the production of endocannabinoid and related lipids through the use of complementary pharmacologic and genetic approaches to disrupt this enzyme in male mice. Here we show that DAGL-α gene deletion or pharmacological inhibition disrupts LTP in CA1 of the hippocampus but elicits varying magnitudes of behavioral learning and memory deficits in mice. In particular, DAGL-α^-/- mice display profound impairments in the Object Location assay and Morris Water Maze (MWM) acquisition engaging in nonspatial search strategies. In contrast, WT mice administered the DAGL-α inhibitor DO34 show delays in MWM acquisition and reversal learning, but no deficits in expression, extinction, forgetting, or perseveration processes in this task, as well as no impairment in Object Location. The deficits in synaptic plasticity and MWM performance occur in concert with decreased 2-AG and its major lipid metabolite (arachidonic acid), but increases of a 2-AG diacylglycerol precursor in hippocampus, PFC, striatum, and cerebellum. These novel behavioral and electrophysiological results implicate a direct and perhaps selective role of DAGL-α in the integration of new spatial information.SIGNIFICANCE STATEMENT Here we show that genetic deletion or pharmacologic inhibition of diacylglycerol lipase-α (DAGL-α) impairs hippocampal CA1 LTP, differentially disrupts spatial learning and memory performance in Morris water maze (MWM) and Object Location tasks, and alters brain levels of endocannabinoids and related lipids. Whereas DAGL-α^-/- mice exhibit profound phenotypic spatial memory deficits, a DAGL inhibitor selectively impairs the integration of new information in MWM acquisition and reversal tasks, but not memory processes of expression, extinction, forgetting, or perseveration, and does not affect performance in the Objection Location task. The findings that constitutive or short-term DAGL-α disruption impairs learning and memory at electrophysiological and selective in vivo levels implicate this enzyme as playing a key role in the integration of new spatial information.

2-Arachidonoylglycerol-mediated endocannabinoid signaling modulates mechanical hypersensitivity associated with alcohol withdrawal in mice

Background: Alcohol use disorder (AUD) commonly occurs in patients with chronic pain, and a major barrier to achieving abstinence and preventing relapse is the emergence of hyperalgesia during alcohol withdrawal. Elucidating novel therapeutic approaches to target hyperalgesia associated with alcohol withdrawal could have important implications for treating AUD. Here, we examined the role of 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid (eCB) signaling in the regulation of hyperalgesia associated with alcohol withdrawal in mice. We tested the hypothesis that pharmacological augmentation of 2-AG signaling could reduce hyperalgesia during withdrawal.
Methods: Male and female C57BL/6J mice were tested during withdrawal from a continuous access two-bottle choice (2BC) paradigm to investigate how eCB signaling modulates mechanical and thermal sensitivity during withdrawal. Mice were pretreated with the monoacylglycerol lipase (MAGL) inhibitor JZL184 to elevate levels of 2-AG. Rimonabant or AM630 were given to block CB₁ and CB₂ receptor activity, respectively. DO34 was given to reduce 2-AG by inhibiting the 2-AG synthetic enzyme diacylglycerol lipase (DAGL).
Results: After 72 h of withdrawal, male and female mice exhibited increased mechanical, but not thermal, hypersensitivity, which normalized by 7 days. This effect was reversed by pretreatment with JZL184. The effects of JZL184 were prevented by coadministration of either the CB₁ or the CB₂ antagonist. DO34, Rimonabant, and AM630 exacerbated mechanical hypersensitivity during alcohol withdrawal, causing an earlier onset and persistent hypersensitivity even 1 week into withdrawal.
Conclusions: Our findings demonstrate the critical role of 2-AG signaling in the bidirectional regulation of mechanical sensitivity during alcohol withdrawal, with enhancement of 2-AG levels reducing sensitivity, and inhibition of 2-AG signaling exacerbating sensitivity. These data suggest that 2-AG augmentation represents a novel approach to the treatment of alcohol withdrawal-associated hyperalgesia and AUD in patients with comorbid pain disorders.

Hypertrophy of human embryonic stem cell-derived cardiomyocytes supported by positive feedback between Ca2+ and diacylglycerol signals

Human embryonic stem cell-derived cardiomyocytes develop pronounced hypertrophy in response to angiotensin-2, endothelin-1, and a selected mix of three fatty acids. All three of these responses are accompanied by increases in both basal cytoplasmic Ca²⁺ and diacylglycerol, quantified with the Ca²⁺ sensor Fluo-4 and a FRET-based diacylglycerol sensor expressed in these cardiomyocytes. The heart glycoside, ouabain (30 nM), and a recently developed inhibitor of diacylglycerol lipases, DO34 (1 μM), cause similar hypertrophy responses, and both responses are accompanied by equivalent increases of basal Ca²⁺ and diacylglycerol. These results together suggest that basal Ca²⁺ and diacylglycerol form a positive feedback signaling loop that promotes execution of cardiac growth programs in these human myocytes. Given that basal Ca²⁺ in myocytes depends strongly on the Na⁺ gradient, we also tested whether nanomolar ouabain concentrations might stimulate Na⁺/K⁺ pumps, as described by others, and thereby prevent hypertrophy. However, stimulatory effects of nanomolar ouabain (1.5 nM) were not verified on Na⁺/K⁺ pump currents in stem cell-derived myocytes, nor did nanomolar ouabain block hypertrophy induced by endothelin-1. Thus, low-dose ouabain is not a "protective" intervention under the conditions of these experiments in this human myocyte model. To summarize, the major aim of this study has been to characterize the progression of hypertrophy in human embryonic stem cell-derived cardiac myocytes in dependence on diacylglycerol and Na⁺ gradient changes, developing a case that positive feedback coupling between these mechanisms plays an important role in the initiation of hypertrophy programs.