Stimulus-Dependent Regulation of 2-Arachidonoyl Glycerol Signaling and the Mechanism of its Hydrolysis by ABHD6

Thesis (Ph.D.)--University of Washington, 2023Cannabis has been used for thousands of years for recreational and therapeutic effects. The efficacy of medicinal cannabis for conditions such as epilepsy and pain has resulted in the discovery of the molecular targets of the bioactive compounds, or phyto-cannabinoids, found in cannabis. These targets are part of a signaling system called the endocannabinoid (eCB) system, which is a neuromodulatory system consisting of the receptors targeted by phyto-cannabinoids, the endogenously produced lipid neuromodulators, or eCBs, that act at these targets, and the machinery that regulates eCB levels, including biosynthetic and degradative enzymes. Enhancing eCB signaling in the brain can provide therapeutic benefit for neurological diseases like epilepsy and chronic pain.A strategy for enhancing eCB signaling is by inhibiting its enzymatic hydrolysis. One of the eCB hydrolyzing enzymes expressed in the brain is ɑ/β hydrolase domain containing 6 (ABHD6). ABHD6 hydrolyzes the eCB 2-arachidonoyl glycerol (2-AG), which is the most abundant eCB in the central nervous system. Production of neuronal 2-AG is activity-dependent, resulting in localized and tightly regulated signaling at targets such as the cannabinoid receptors (CBRs). Therefore, inhibiting ABHD6 can increase intrinsic 2-AG levels and resulting CBR activation. Thus, inhibition will have an effect only when and where activated cells produce 2-AG. Recent studies have demonstrated efficacy of ABHD6 inhibition in preclinical models of epilepsy and neuropathic pain, which are pathological conditions that share key elements: hyperexcitability of neurons and neuroinflammation. While 2-AG’s role in reducing neuronal activity has been well-described, the mechanism of neuronal 2-AG production following inflammation and the effect of ABHD6 inhibition on enhancing these 2-AG levels remains unclear. Here, we characterize a genetically encoded sensor, the GRABeCB2.0, and then use the sensor to show that two inflammatory mediators, ATP and bradykinin (BK), can stimulate 2-AG production in neuro2a mouse neuroblastoma cells through separate receptor-dependent mechanisms. I discovered that, in a co-culture model system, ATP and BK-stimulated 2-AG can participate in paracrine activation of GRABeCB2.0. These findings describe a potential molecular mechanism by which inflammatory mediators may act directly on sensory CB1R-expressing neurons and engage the eCB system, which can function in a negative feedback loop to decrease hyperactivity of peripheral nociceptors that underlies hyperalgesia. Since ABHD6 inhibition has the potential to enhance 2-AG signaling and produces analgesia in neuropathic pain models, we further described the mechanism of ABHD6 hydrolysis activity and discovered that an undescribed membrane factor increases in vitro ABHD6 activity and are now testing a first-in-class ABHD6 inhibitor. This work expands our understanding of the pathophysiological role of 2-AG and describes the function of ABHD6, a potential therapeutic target which can allow for the development selective inhibitors with a clinical benefit