∆9-tetrahydrocannabinol negatively regulates neurite outgrowth and Akt signaling in hiPSC-derived cortical neurons

Endocannabinoids regulate different aspects of neurodevelopment. In utero exposure to the exogenous psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC), has been linked with abnormal cortical development in animal models. However, much less is known about the actions of endocannabinoids in human neurons. Here we investigated the effect of the endogenous endocannabinoid 2-arachidonoyl glycerol (2AG) and Δ9-THC on the development of neuronal morphology and activation of signaling kinases, in cortical glutamatergic neurons derived from human induced pluripotent stem cells (hiPSCs). Our data indicate that the cannabinoid type 1 receptor (CB1R), but not the cannabinoid 2 receptor (CB2R), GPR55 or TRPV1 receptors, is expressed in young, immature hiPSC-derived cortical neurons. Consistent with previous reports, 2AG and Δ9-THC negatively regulated neurite outgrowth. Interestingly, acute exposure to both 2AG and Δ9-THC inhibited phosphorylation of serine/threonine kinase extracellular signal-regulated protein kinases (ERK1/2), whereas Δ9-THC also reduced phosphorylation of Akt (aka PKB). Moreover, the CB1R inverse agonist SR 141716A attenuated the negative regulation of neurite outgrowth and ERK1/2 phosphorylation induced by 2AG and Δ9-THC. Taken together, our data suggest that hiPSC-derived cortical neurons express CB1Rs and are responsive to both endogenous and exogenous cannabinoids. Thus, hiPSC-neurons may represent a good cellular model for investigating the role of the endocannabinoid system in regulating cellular processes in human neurons.


Introduction
The endocannabinoid system is a neuromodulatory system with important roles in central nervous system (CNS) development, neuronal function and synaptic plasticity 1,2 . Perturbations in this system have been observed in psychiatric disorders such as schizophrenia 3 , and there is a significant association between cannabis use and schizophrenia [3][4][5][6] . The endocannabinoid system is composed of endogenous cannabinoids, enzymes that synthesize and degrade endogenous cannabinoids and cannabinoid receptors. The Cannabinoid 1 receptor (CB1R) is a highly abundant receptor in the CNS, with strong expression in a number of brain regions, including the cortex 1,7 . Cannabinoid 2 receptors (CB2Rs) show much lower expression in the CNS, although recent work has shown high inducible expression of CB2Rs under pathological conditions 8 . Several other receptors, such as peroxisome proliferator activated receptors and transient receptor potential channels, are also engaged by cannabinoids 9 .
CB1Rs are members of the superfamily of G protein-coupled receptors that inhibit adenylyl cyclase and activate mitogen-activated protein kinase by signaling through G i/o proteins. Stimulation of CB1R by cannabinoids has been shown to lead range of effects including a depression of the glutamatergic system 1 . This highlights the potential importance of the CB1R in relation to health and disease.
Much of our knowledge of the endocannabinoid system comes from work using in vitro and in vivo animal models. It remains unknown how well these data translate to the human neurons. Recent investigations into the role of endocannabinoids during cortical development have shown that cannabinoids regulate neuronal morphology, through small GTPase signaling pathways and rearrangement of the actin cytoskeleton, resulting in a negative impact on cortical development [13][14][15] . Recent advances in stem cell technology have enabled the reprogramming of human somatic cells into induced pluripotent stem cells (hiPSCs) 16,17 , which can subsequently be differentiated in to specific neuronal subtypes 18 . Using this system Guennewig and colleagues demonstrated that exposure of hiPSC-neurons to Δ 9 -THC resulted in significant alterations in genes involved with development, synaptic function, as well as those associated with psychiatric disorders 19 . This supports the use of hiPSCs as a cellular model to investigate the cellular actions of cannabinoids to understand their relevance for health and disease.
In this study, we first examined the expression of cannabinoid receptors during the differentiation of hiPSCs into cortical glutamatergic neurons.
Subsequently, we investigated the impact of acute exposure to the endogenous cannabinoid 2-arachidonoyl glycerol (2AG), a full agonist for the CB1R, and  Taken together, our data indicates that CB1R is expressed in immature iPSCneurons and that 2AG and Δ 9 -THC treatment negatively affect neuronal morphology and impact the Akt and ERK1/2 signaling pathways, potentially via CB1R. These data indicate that cannabinoids may act to influence the development of human cortical neurons.

Generation of cortical neurons from hiPSCs
Within the developing CNS, the endocannabinoid system has been implicated in important neuronal processes such as synapse formation and neurogenesis 1,2 .
CB1Rs are G-protein coupled receptors that are widely expressed in the brain 7 , including the cortex 1,2 . Consistent with a role for the endocannabinoid system during the development of cortical neurons, acute (24 hour) exposure of hiPSC-neurons to Δ 9 -THC results in the differential expression of multiple genes, including those involved in development 19 . Therefore, in order to further understand the role of cannabinoids during the development of human cortical neurons, we induced cortical differentiation from hiPSC. The cell lines were derived by reprogramming keratinocytes from 3 neurotypic males (age ranging between 35 to 55 years: CTM_01_04, CTM_02_05 and CTM_03_22) [20][21][22][23] . HiPSC lines were differentiated into neuroepithelial cell using a dual SMAD inhibition (2i) differentiation protocol for 8 days 20,21,23 ( Figure 1A). The small molecules inhibitors were removed during neuronal progenitor's differentiation before media was replaced with B27 supplemented with DAPT to induce the generation of terminally differentiated neurons 20,21,23 (Figure 1A). Immunocytochemical (ICC) analyses indicated that the derived hiPSC expressed high levels of the pluripotency markers OCT4, NANOG and SOX11, whereas early neural progenitor cell (NPC) markers PAX6, ZNF521 and NESTIN were up-regulated following 8 days of 2i induction (Figure 1B and 1C).
After 26 days of neuronal differentiation young neurons expressed high levels of FOXG1, TBR1, TBR2 and BRN2 factors, which are required for the differentiation of cortical neurons (Figure 1B and 1C). Similarly, real time PCR (qPCR) analyses demonstrated that the neuroepithelial marker SOX2 was highly expressed 7 days after 2i induction and was gradually down-regulated during the 50 days of neuronal fate acquisition ( Figure 1D). PAX6, a key regulator of cortical development, was found to be highly expressed from day 7 to day 50 during neuronal formation; peak expression was observed at day 22 ( Figure 1E). The expression of CTIP2, a marker of deep layer cortical cell fate, rapidly increased from day 22 to day 28, peaking at day 26; subsequently, expression of CTIP2 was down-regulated until day 50 ( Figure   1F). Conversely, expression of BRN2, a marker associated with upper cortical layer cell fate, showed expression peaks at days 23, 26 and once more at day 48 ( Figure   1G). At day 30, no GFAP or S100β positive cells were observed (Supplementary Figure 1). ICC analysis at day 50 revealed that most MAP-positive cells were also immunoreactive for markers of glutamatergic fate. This included the -presynaptic proteins synapsin 1 and VGlut1 and the post-synaptic proteins PSD95 and GluN1subunit of NMDA receptors (Figure 2A-C). To confirm that hiPSC-neurons generated from a hiPSC line derived from keratinocytes, and following our 2i cortical protocol, develop physiological neuronal characteristics, we conducted whole-cell patch clamp recordings. Current clamp recordings demonstrated that these cells were capable of firing an action potential in response to current injection ( Figure 2E).
Depolarising step potentials (from a holding potential of -60mV) evoked transient inward currents and sustained outward currents, indicative of activation of voltage gated sodium channels and potassium channels respectively (Figure 2E). At this age, we were able to record isolated spontaneous excitatory postsynaptic currents (EPSCs), demonstrating the presence of functional excitatory synaptic connections at this stage ( Figure 2F). Collectively, these data demonstrate the generation of cortical glutamatergic neurons from hiPSCs.

CB1R expression during the cortical differentiation of iPSCs
Previous studies have suggested that the CB1R is highly expressed during neuronal development 1 . Therefore, we examined the expression of CB1R, at the mRNA level, in hiPSC-derived NPCs and terminally differentiated hiPSC-derived cortical neurons. In all three hiPSC lines, CB1R showed similar gene expression patterns across the three iPSC lines. Specifically, CB1R mRNA levels significantly increased as hiPSCs differentiated from NPCs into neurons ( Figure 3A). We also examined the expression of CB2R, GPR55 and TRPV1 receptors which are expressed in the brain and are engaged by cannabinoids in differentiated neurons 1,2 .
The mRNA expression levels of these receptors were mirrored across the three hiPSC lines and was significantly reduced compared with the expression of CB1R ( Figure 3B). Next, we examined the expression of CB1R in immature hiPSCneurons using an antibody raised against the C-terminal of the receptor and validated in knockout tissue and human tissue 24,25 as well by pre-absorption studies (Supplemental Figure 2A). Western blotting demonstrated that the receptor was expressed at the protein level in immature hiPSC-neurons. A prominent band ~53 kDa consistent with the predicated molecular weight for the receptor, was readily observed ( Figure 3C). Consistent with these data, immunostaining of immature hiPSC-neurons revealed that CB1R staining could be observed in all three hiPSC clonal lines. CB1R immunoreactivity could be observed within the cell soma, as well as punctate structures along MAP2-positive neurites ( Figure 3D). Collectivity, these data demonstrate that CB1R is expressed in immature hiPSC-neurons and is ideally localized to influence neuronal morphology as previously reported. There is growing appreciation that the endocannabinoid system is an important regulator of brain wiring during development through the modulation of several different processes including the specification of neuronal morphology 13,14,[26][27][28] . As CB1R is localized along neurite, we reasoned that endocannabinoids may be involved in regulating the establishment of neuronal morphology in hiPSC-neurons.
To test this prediction, we first treated hiPSC-neurons at day 29 with 1 µM 2AG, a full agonist for the CB1R, for 24 hours. Neurons were then fixed and stained for MAP2 to outline neuronal morphology. Neurite outgrowth was assayed using a high-content screening platform ( Figure 4A). We first assessed whether treatment with 2AG  9 -THC has previously been shown to negatively regulate neurite outgrowth and growth cone dynamics, through the regulation of actin polymerization and microtubule stability in mouse cortical neurons 13,15,26 . In addition, exposure to Δ 9 -THC in hiPSC-neurons results in the alteration of multiple genes involved in the development of neuronal morphology 19 . Therefore, we next examined whether acute exposure to       Stimulation of CB1R leads to the phosphorylation and activation of several signaling kinases, including Akt, ERk1/2 and GSK3β 2,29,30 . Akt and ERK1/2 pathways have also been implicated in promoting neurite outgrowth 11,12 . GSK-3β, a welldefined substrate of Akt, has also been implicated in the regulation of neurite and axonal outgrowth and branching 31 . Interestingly, the negative effects of CB1R activation on neurite outgrowth have been linked with ERK1/2 signaling 19 , although activation of CB1R has also been linked with Akt/GSK3β signaling in vivo 30 . Therefore, we first examined 2AG generated changes in the activation state (phosphorylation) of ERK1/2 and Akt/GSK3β kinases in day 30 hiPSC-neurons. 2AG treatment resulted in a significant decrease in ERK1/2 phosphorylation after 30 minutes ( Figure 6A). Conversely, no changes in the phosphorylation state of Akt or GSK3β were detected following 15 and 30 minutes of 2AG exposure (Figure 6B and C). These data demonstrated that 2AG, a full agonist for the CB1R, is capable of negatively regulating ERK1/2 activity.
Owing to our data demonstrating that 2AG negatively regulated ERK1/2 phosphorylation, we next investigated the effect of   -THC also caused a rapid reduction in levels of phosphorylated Akt, which was evident after 15 minutes ( Figure 6E). A non-significant trend towards a decrease in phosphorylated GSK3β was also evident following Δ 9 -THC treatment, but this did not reach significance ( Figure 6F; p=0.105). Collectively, these data indicate that Δ 9 -THC negatively regulates both ERK1/2 and Akt phosphorylation, in contrast to 2AG, which regulates ERK1/2 exclusively.

-THC modulate Akt and ERK1/2 phosphorylation via the CB1R receptor.
As our data indicated that both 2AG and    these data indicate that 2AG and Δ 9 -THC negatively regulated ERK1/2 and Akt signaling kinases by acting through the CB1R receptor. As our data indicated that the CB1R mediates the effects of 2AG and Δ 9 -THC on ERK1/2 activity, we next questioned whether this receptor was required for the negative regulation of neurite outgrowth y these cannabinoids. To test this, we treated day 29 hiPSC-neurons with 2AG or Δ 9 -THC with or without SR 141716A.

Negative regulation of neurite outgrowth by 2AG and
Treatment with 2AG or Δ 9 -THC caused a reduction in total neurite length; this was attenuated by co-treatment with SR 141716A (Figure 7A

Discussion
Recently it has been demonstrated that cannabinoid treatment affects neuronal function and glutamate receptor expression in hiPSC-derived dopaminergic neurons and excitatory neurons, respectively 32,33 . Moreover, treatment with      Overall, the results of the present study suggest that young hiPSC-derived glutamatergic neurons are responsive to cannabinoids, and further support the notion that this cellular system is a good model for investigating CB1R signaling 19 .
CB1Rs were first identified as the main neuronal receptor for Δ 9 -THC and are one of the most abundant G protein-coupled receptors in the brain 7 . These receptors have previously been characterized in being involved in retrograde synaptic signaling, acting to control neuronal activity in mature neurons [41][42][43] and to affect both long-term potentiation (LTP) and depression (LTD) and to impair learning and memory 44 .
CB1Rs and endocannabinoids are also highly expressed in the fetal brain and implicated in neuronal development processes such as neurite growth and axonal pathfinding 13,15,[26][27][28]35 . As shown in this and other recent studies, both endogenous and exogenous cannabinoids alter neurite outgrowth, synaptic activity and synaptic protein expression in developing human hiPSC-derived neurons 32,33 . Moreover, as Δ 9 -THC has recently been shown to alter expression of genes associated with neurodevelopmental and psychiatric disorders, there is evidence for perturbations of shared molecular pathways potentially exacerbated by Δ 9 -THC 19 . Thus, these data collectively suggest that cannabinoids may play a role in brain development.
Furthermore, they raise the possibility that overstimulation by exogenous cannabinoids or abnormal levels of endocannabinoids may perturb normal physiological processes, during neurodevelopment which may contribute to the pathophysiology of neurodevelopmental and psychiatric disorders, however further in-depth studies are required to investigate these possibilities.   Table 1.

Human induced pluripotent stem cells (hiPSCs)
hiPSC lines were generated from primary keratinocytes as described previously 20

Neuronal differentiation
Neuronal differentiation of hiPSCs was by achieved replacing E8 medium on

Imaging of immunofluorescence by high content image screening
NPCs were plated at a density of 1x10 4

Competing Interests
Authors declare no conflict of interest