A Complete Endocannabinoid Signaling System Modulates Synaptic Transmission between Human Induced Pluripotent Stem Cell-Derived Neurons

Melissa J Asher; Hannah M McMullan; Ao Dong; Yulong Li; Stanley A. Thayer

doi:10.1124/molpharm.122.000555

A Complete Endocannabinoid Signaling System Modulates Synaptic Transmission between Human Induced Pluripotent Stem Cell-Derived Neurons

Melissa J Asher, Hannah M McMullan, Ao Dong, Yulong Li, Stanley A. Thayer

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Abstract

The endocannabinoid system (ECS) modulates synaptic function to regulate many aspects of neurophysiology. It adapts to environmental changes and is affected by disease. Thus, the ECS presents an important target for therapeutic development. Despite recent interest in cannabinoid-based treatments, few preclinical studies are conducted in human systems. Human induced pluripotent stem cells (hiPSCs) provide one possible solution to this issue. However, it is not known if these cells have a fully functional ECS. Here, we show that hiPSC-derived neuron/astrocyte cultures exhibit a complete ECS. Using Ca2+ imaging and a genetically encoded endocannabinoid sensor, we demonstrate that they not only respond to exogenously applied cannabinoids but also produce and metabolize endocannabinoids. Synaptically driven [Ca2+]i spiking activity was inhibited (EC505 48 ± 13 nM) by the efficacious agonist [R(1)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] (Win 55,212-2) and by the endogenous ligand 2-arachidonoyl glycerol (2-AG; EC50 5 2.0 ± 0.6 mm). The effects of Win 55212-2 were blocked by a CB1 receptor-selective antagonist. D9-Tetrahydrocannabinol acted as a partial agonist, maximally inhibiting synaptic activity by 47 ± 14% (EC50 5 1.4 ± 1.9 mm). Carbachol stimulated 2-AG production in a manner that was independent of Ca2+ and blocked by selective inhibition of diacylglycerol lipase. 2-AG returned to basal levels via a process mediated bymonoacylglycerol lipase as indicated by slowed recovery in cultures treated with 4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester (JZL 184). Win 55,212-2 markedly desensitized CB1 receptor function following a 1-day exposure, whereas desensitization was incomplete following 7-day treatment with JZL 184. This human cell culture model is well suited for functional analysis of the ECS and as a platformfor drug development.

Original language	English (US)
Pages (from-to)	100-112
Number of pages	13
Journal	Molecular Pharmacology
Volume	103
Issue number	2
DOIs	https://doi.org/10.1124/molpharm.122.000555
State	Published - Feb 1 2023

Bibliographical note

Funding Information:
This work was made possible by National Institutes of Health grants from the National Institute on Drug Abuse [Grant DA07304] (to S.A.T.) and the National Institute of Mental Health [Grant MH122193] (to S.A.T.) and support from the Viral Vector and Cloning Core/Viral Innovation Core [Grant P30 DA048742-01A1].

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© 2023 by The American Society for Pharmacology and Experimental Therapeutics.

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title = "A Complete Endocannabinoid Signaling System Modulates Synaptic Transmission between Human Induced Pluripotent Stem Cell-Derived Neurons",

abstract = "The endocannabinoid system (ECS) modulates synaptic function to regulate many aspects of neurophysiology. It adapts to environmental changes and is affected by disease. Thus, the ECS presents an important target for therapeutic development. Despite recent interest in cannabinoid-based treatments, few preclinical studies are conducted in human systems. Human induced pluripotent stem cells (hiPSCs) provide one possible solution to this issue. However, it is not known if these cells have a fully functional ECS. Here, we show that hiPSC-derived neuron/astrocyte cultures exhibit a complete ECS. Using Ca2+ imaging and a genetically encoded endocannabinoid sensor, we demonstrate that they not only respond to exogenously applied cannabinoids but also produce and metabolize endocannabinoids. Synaptically driven [Ca2+]i spiking activity was inhibited (EC505 48 ± 13 nM) by the efficacious agonist [R(1)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] (Win 55,212-2) and by the endogenous ligand 2-arachidonoyl glycerol (2-AG; EC50 5 2.0 ± 0.6 mm). The effects of Win 55212-2 were blocked by a CB1 receptor-selective antagonist. D9-Tetrahydrocannabinol acted as a partial agonist, maximally inhibiting synaptic activity by 47 ± 14% (EC50 5 1.4 ± 1.9 mm). Carbachol stimulated 2-AG production in a manner that was independent of Ca2+ and blocked by selective inhibition of diacylglycerol lipase. 2-AG returned to basal levels via a process mediated bymonoacylglycerol lipase as indicated by slowed recovery in cultures treated with 4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester (JZL 184). Win 55,212-2 markedly desensitized CB1 receptor function following a 1-day exposure, whereas desensitization was incomplete following 7-day treatment with JZL 184. This human cell culture model is well suited for functional analysis of the ECS and as a platformfor drug development.",

author = "Asher, {Melissa J} and McMullan, {Hannah M} and Ao Dong and Yulong Li and Thayer, {Stanley A.}",

note = "Funding Information: This work was made possible by National Institutes of Health grants from the National Institute on Drug Abuse [Grant DA07304] (to S.A.T.) and the National Institute of Mental Health [Grant MH122193] (to S.A.T.) and support from the Viral Vector and Cloning Core/Viral Innovation Core [Grant P30 DA048742-01A1]. Publisher Copyright: {\textcopyright} 2023 by The American Society for Pharmacology and Experimental Therapeutics.",

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AU - Thayer, Stanley A.

N1 - Funding Information: This work was made possible by National Institutes of Health grants from the National Institute on Drug Abuse [Grant DA07304] (to S.A.T.) and the National Institute of Mental Health [Grant MH122193] (to S.A.T.) and support from the Viral Vector and Cloning Core/Viral Innovation Core [Grant P30 DA048742-01A1]. Publisher Copyright: © 2023 by The American Society for Pharmacology and Experimental Therapeutics.

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N2 - The endocannabinoid system (ECS) modulates synaptic function to regulate many aspects of neurophysiology. It adapts to environmental changes and is affected by disease. Thus, the ECS presents an important target for therapeutic development. Despite recent interest in cannabinoid-based treatments, few preclinical studies are conducted in human systems. Human induced pluripotent stem cells (hiPSCs) provide one possible solution to this issue. However, it is not known if these cells have a fully functional ECS. Here, we show that hiPSC-derived neuron/astrocyte cultures exhibit a complete ECS. Using Ca2+ imaging and a genetically encoded endocannabinoid sensor, we demonstrate that they not only respond to exogenously applied cannabinoids but also produce and metabolize endocannabinoids. Synaptically driven [Ca2+]i spiking activity was inhibited (EC505 48 ± 13 nM) by the efficacious agonist [R(1)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] (Win 55,212-2) and by the endogenous ligand 2-arachidonoyl glycerol (2-AG; EC50 5 2.0 ± 0.6 mm). The effects of Win 55212-2 were blocked by a CB1 receptor-selective antagonist. D9-Tetrahydrocannabinol acted as a partial agonist, maximally inhibiting synaptic activity by 47 ± 14% (EC50 5 1.4 ± 1.9 mm). Carbachol stimulated 2-AG production in a manner that was independent of Ca2+ and blocked by selective inhibition of diacylglycerol lipase. 2-AG returned to basal levels via a process mediated bymonoacylglycerol lipase as indicated by slowed recovery in cultures treated with 4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester (JZL 184). Win 55,212-2 markedly desensitized CB1 receptor function following a 1-day exposure, whereas desensitization was incomplete following 7-day treatment with JZL 184. This human cell culture model is well suited for functional analysis of the ECS and as a platformfor drug development.

AB - The endocannabinoid system (ECS) modulates synaptic function to regulate many aspects of neurophysiology. It adapts to environmental changes and is affected by disease. Thus, the ECS presents an important target for therapeutic development. Despite recent interest in cannabinoid-based treatments, few preclinical studies are conducted in human systems. Human induced pluripotent stem cells (hiPSCs) provide one possible solution to this issue. However, it is not known if these cells have a fully functional ECS. Here, we show that hiPSC-derived neuron/astrocyte cultures exhibit a complete ECS. Using Ca2+ imaging and a genetically encoded endocannabinoid sensor, we demonstrate that they not only respond to exogenously applied cannabinoids but also produce and metabolize endocannabinoids. Synaptically driven [Ca2+]i spiking activity was inhibited (EC505 48 ± 13 nM) by the efficacious agonist [R(1)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] (Win 55,212-2) and by the endogenous ligand 2-arachidonoyl glycerol (2-AG; EC50 5 2.0 ± 0.6 mm). The effects of Win 55212-2 were blocked by a CB1 receptor-selective antagonist. D9-Tetrahydrocannabinol acted as a partial agonist, maximally inhibiting synaptic activity by 47 ± 14% (EC50 5 1.4 ± 1.9 mm). Carbachol stimulated 2-AG production in a manner that was independent of Ca2+ and blocked by selective inhibition of diacylglycerol lipase. 2-AG returned to basal levels via a process mediated bymonoacylglycerol lipase as indicated by slowed recovery in cultures treated with 4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester (JZL 184). Win 55,212-2 markedly desensitized CB1 receptor function following a 1-day exposure, whereas desensitization was incomplete following 7-day treatment with JZL 184. This human cell culture model is well suited for functional analysis of the ECS and as a platformfor drug development.

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A Complete Endocannabinoid Signaling System Modulates Synaptic Transmission between Human Induced Pluripotent Stem Cell-Derived Neurons

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