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Molecular model of cannabis sensitivity in developing neuronal circuits

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Prenatal cannabis exposure can complicate in utero development of the nervous system. Cannabis impacts the formation and functions of neuronal circuitries by targeting cannabinoid receptors. Endocannabinoid signaling emerges as a signaling cassette that orchestrates neuronal differentiation programs through the precisely timed interaction of endocannabinoid ligands with their cognate cannabinoid receptors. By indiscriminately prolonging the ‘switched-on’ period of cannabinoid receptors, cannabis can hijack endocannabinoid signals to evoke molecular rearrangements, leading to the erroneous wiring of neuronal networks. Here, we formulate a hierarchical network design necessary and sufficient to describe the molecular underpinnings of cannabis-induced neural growth defects. We integrate signalosome components, deduced from genome- and proteome-wide arrays and candidate analyses, to propose a mechanistic hypothesis of how cannabis-induced ectopic cannabinoid receptor activity overrides physiological neurodevelopmental endocannabinoid signals, affecting the timely formation of synapses.

Section snippets

Endocannabinoids: gatekeepers of neuronal development

Molecular cloning of the CB1 cannabinoid receptor (CB1R) [1] and its functional characterization as the major target of Δ9-tetrahydrocannabinol (THC) from cannabis [2] led to a sea-change in the understanding of the molecular mechanisms of this psychoactive drug's actions on neuronal structure and function in brain regions controlling memory, cognition, movement and pain perception [3]. These findings, coupled with the discovery that the CB1R functions as an essential signal transducer in an

The ‘high’-way of brain development: clinical considerations

THC can enter the fetal circulation with rapid onset [25] via efficient transfer through the placenta [26]. THC levels in the amniotic fluid and fetus remain elevated for up to 5 h, followed by gradual clearance within 48 h after exposure [25]. Exceptionally high THC doses (>100 mg/kg) may be teratogenic and induce in utero death [25]. However, cannabis use during pregnancy can lead to growth retardation [27] and is associated with adverse neurodevelopmental outcomes [28]. The delay in nervous

Endocannabinoids in the nervous system

In addition to the best known ligands, 2-arachidonoyl glycerol (2-AG) 42, 43, 44 and N-arachidonoylethanolamine (AEA) [45], the list of possible endocannabinoids includes an increasing number of structurally related ligands with appreciable pharmacological efficacy at the CB1R or CB2 cannabinoid receptor (as well as having other targets; Box 1, Table 1). However, the concentration, regionalized distribution, and metabolic and signaling interactions of these endocannabinoid-like substances

Molecular organization of endocannabinoid signaling in developing neurons

Neurogenic commitment differentially regulates expression of the CB1R [7], and sn-1-diacylglycerol lipase α and β (DAGLα/β) [11] and monoacylglycerol lipase (MAGL) [50] – 2-AG synthesizing and degrading enzymes, respectively. Neuronal differentiation upregulates CB1R expression [7], whereas cell cycle exit of neural stem cells represses DAGLs [11]. During neuronal polarization, this signaling triad is cotransported along the nascent axon (Figure 1a and b) 6, 13, 15, 16. Although CB1Rs can be

Endocannabinoids: intracellular or extracellular signals?

Endocannabinoids are viewed as lipophilic ligands, the ability of which to disperse in an aqueous extracellular environment may be limited. However, this view is challenged by the absence of retrograde synaptic signaling in DAGLα−/− mice 18, 55. Because retrograde signaling relies on facilitated transsynaptic 2-AG diffusion to activate presynaptic CB1Rs, data from DAGLα−/− models unequivocally identify 2-AG as an extracellular retrograde messenger. 2-AG can also signal intracellularly [46].

Is endocannabinoid signaling indispensable for brain development?

The answer to this question might be ambiguous for several reasons. First, developmentally redundant signaling cassettes rely on promiscuous ligand–receptor interactions to sustain signaling efficacy, even when a component of the signaling system is compromised. Therefore, the use of constitutive and global gene knockouts to determine the function of a single gene may be limited in deciphering the absolute contribution of a candidate mechanism to homeostatic control pathways 18, 55, 56. Second,

CB1R can diversify the ontogenic impact of endocannabinoid signals

The CB1R exhibits tremendous signaling complexity. The simplest functional unit of a G protein-coupled receptor (GPCR) (including the CB1R) is a homodimer that recruits a heterotrimeric G protein [59]. However, the CB1R is physiologically ‘dominant’ because it can be coupled to Gi/o proteins even in the absence of an agonist (‘constitutive activity’), thereby depleting the common intracellular Gi/o pool and limiting the biological signals of other Gi/o-coupled receptors [60]. GPCRs can also

Signaling pathways linking the CB1R to cell proliferation/survival and neurite outgrowth

Classical Gi protein-mediated signaling at the CB1R is well suited to activate effectors that couple endocannabinoid signaling to cell survival, proliferation and differentiation (Figure 2). Here, we discuss novel facets of the understanding of cell state-specific signaling events that have evolved since a recent survey of available data [62] and emphasize the importance of CB1R signaling in relation to axonal growth and guidance 15, 17, 71, 72.

A major branch of CB1R signaling is directed

Receptor interactions sensitizing developing neurons to endocannabinoids

Molecular arrangements exerting upstream control on endocannabinoid signaling during neurite outgrowth are essential in defining the ultimate physiological outcome. An appealing hypothesis implicates Trks (e.g. fibroblast growth factor receptor) because they induce Ca2+ mobilization through PLCγ [78]. Elevated intracellular Ca2+ can activate DAGLs to generate 2-AG and trigger CB1R activation [24]. The robustness of Trk signaling is epitomized by: i) the ability of brain-derived neurotrophic

Compartmentalized signaling by the CB1R

Any instructive signal must be restricted spatially to encode positional information. This can be achieved by limiting the expression, cell surface presentation and extracellular spread of a ligand or by compartmentalizing ligand–receptor interactions. Neurotrophin receptors, DCC and probably the CB1R can be clustered by function as ‘dependence’ (or survival) receptors because they create cellular states of dependence towards their cognate ligands by inducing apoptosis if left unoccupied [81].

THC can hijack physiologically silent CB1Rs

In developing cortical axons, MAGL forms a 2-AG-inactivating barrier in the established axon segment, and can function as a ‘switch-off system’ to terminate 2-AG signaling (Figure 3a) [13]. We postulate that the role of MAGL in elongating axons is to prevent lipophilic 2-AG from accessing and prematurely engaging intracellular CB1Rs in the axon stem as they undergo axonal transport.

The differential recruitment of DAGLs and MAGL along the axon might serve several independent modes of

The molecular fingerprint of prenatal cannabis abuse

If THC affects developmental processes, its molecular fingerprint must involve developmentally regulated genes. The cumulative complexity of this gene/protein network will reflect the relative ‘power’ of THC to affect developmental processes. Global genome and proteome profiling after exposure to THC or CB1R agonists in utero (or during adolescence) increasingly support this notion by identifying a largely invariable cluster of target molecules (Figure 4).

Disrupting the temporal precision of CB1

Concluding remarks

Whether THC is an agonist or antagonist at the CB1R during development, when neurons require a high intrinsic endocannabinoid tone to sustain growth processes, is debated [24]. Nevertheless, the concept that THC exerts its detrimental effects by disrupting the temporal and spatial cohesion of endocannabinoid signaling has recently gained momentum 12, 14, 24. Based on the evidence from cell and systems biology presented here, we conclude that the functional redundancy of the many endocannabinoid

Acknowledgements

The authors thank P. Doherty and R.A. Ross for constructive criticism and discussions. This work was supported by the Scottish Universities Life Science Alliance (T.H.), the Swedish Medical Research Council (T.H.), the European Commission (HEALTH-F2-2007-201159, T.H.) and the National Institutes of Health grants DA023214 (T.H.), DA11322 (K.M.) and DA021696 (K.M).

Glossary

Cell cycle exit
the event when a cell permanently leaves the cell cycle to adopt a terminal differentiation program. To achieve this, cells become refractory to proliferative signals.
Fate decision
the point when a progenitor cell commits toward and initiates an intrinsic specification program to generate a terminally differentiated cell.
Lateral geniculate nucleus
resides within the thalamus and functions as the primary relay center for visual information received from the retina of the eye.
Growth

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