Endocannabinoid signalling in stem cells and cerebral organoids drives differentiation to deep layer projection neurons via CB1 receptors.

The endocannabinoid (eCB) system, via the cannabinoid CB1 receptor, regulates neurodevelopment by controlling neural progenitor proliferation and neurogenesis. CB1 receptor signalling in vivo drives corticofugal deep layer projection neuron development through the regulation of BCL11B and SATB2 transcription factors. Here, we investigated the role of eCB signalling in mouse pluripotent embryonic stem cell-derived neuronal differentiation. Characterization of the eCB system revealed increased expression of eCB-metabolizing enzymes, eCB ligands and CB1 receptors during neuronal differentiation. CB1 receptor knockdown inhibited neuronal differentiation of deep layer neurons and increased upper layer neuron generation, and this phenotype was rescued by CB1 re-expression. Pharmacological regulation with CB1 receptor agonists or elevation of eCB tone with a monoacylglycerol lipase inhibitor promoted neuronal differentiation of deep layer neurons at the expense of upper layer neurons. Patch-clamp analyses revealed that enhancing cannabinoid signalling facilitated neuronal differentiation and functionality. Noteworthy, incubation with CB1 receptor agonists during human iPSC-derived cerebral organoid formation also promoted the expansion of BCL11B+ neurons. These findings unveil a cell-autonomous role of eCB signalling that, via the CB1 receptor, promotes mouse and human deep layer cortical neuron development.

Identification of BiP as a CB1 Receptor-Interacting Protein That Fine-Tunes Cannabinoid Signaling in the Mouse Brain.

Cannabinoids, the bioactive constituents of cannabis, exert a wide array of effects on the brain by engaging Type 1 cannabinoid receptor (CB1R). Accruing evidence supports that cannabinoid action relies on context-dependent factors, such as the biological characteristics of the target cell, suggesting that cell population-intrinsic molecular cues modulate CB1R-dependent signaling. Here, by using a yeast two-hybrid-based high-throughput screening, we identified BiP as a potential CB1R-interacting protein. We next found that CB1R and BiP interact specifically in vitro, and mapped the interaction site within the CB1R C-terminal (intracellular) domain and the BiP C-terminal (substrate-binding) domain-α. BiP selectively shaped agonist-evoked CB1R signaling by blocking an "alternative" Gq/11 protein-dependent signaling module while leaving the "classical" Gi/o protein-dependent inhibition of the cAMP pathway unaffected. In situ proximity ligation assays conducted on brain samples from various genetic mouse models of conditional loss or gain of CB1R expression allowed to map CB1R-BiP complexes selectively on terminals of GABAergic neurons. Behavioral studies using cannabinoid-treated male BiP+/- mice supported that CB1R-BiP complexes modulate cannabinoid-evoked anxiety, one of the most frequent undesired effects of cannabis. Together, by identifying BiP as a CB1R-interacting protein that controls receptor function in a signaling pathway- and neuron population-selective manner, our findings may help to understand the striking context-dependent actions of cannabis in the brain.SIGNIFICANCE STATEMENT Cannabis use is increasing worldwide, so innovative studies aimed to understand its complex mechanism of neurobiological action are warranted. Here, we found that cannabinoid CB1 receptor (CB1R), the primary molecular target of the bioactive constituents of cannabis, interacts specifically with an intracellular protein called BiP. The interaction between CB1R and BiP occurs selectively on terminals of GABAergic (inhibitory) neurons, and induces a remarkable shift in the CB1R-associated signaling profile. Behavioral studies conducted in mice support that CB1R-BiP complexes act as fine-tuners of anxiety, one of the most frequent undesired effects of cannabis use. Our findings open a new conceptual framework to understand the striking context-dependent pharmacological actions of cannabis in the brain.

Δ9 -Tetrahydrocannabinol promotes oligodendrocyte development and CNS myelination in vivo.

Δ9 -Tetrahydrocannabinol (THC), the main bioactive compound found in the plant Cannabis sativa, exerts its effects by activating cannabinoid receptors present in many neural cells. Cannabinoid receptors are also physiologically engaged by endogenous cannabinoid compounds, the so-called endocannabinoids. Specifically, the endocannabinoid 2-arachidonoylglycerol has been highlighted as an important modulator of oligodendrocyte (OL) development at embryonic stages and in animal models of demyelination. However, the potential impact of THC exposure on OL lineage progression during the critical periods of postnatal myelination has never been explored. Here, we show that acute THC administration at early postnatal ages in mice enhanced OL development and CNS myelination in the subcortical white matter by promoting oligodendrocyte precursor cell cycle exit and differentiation. Mechanistically, THC-induced-myelination was mediated by CB1 and CB2 cannabinoid receptors, as demonstrated by the blockade of THC actions by selective receptor antagonists. Moreover, the THC-mediated modulation of oligodendroglial differentiation relied on the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, as mTORC1 pharmacological inhibition prevented the THC effects. Our study identifies THC as an effective pharmacological strategy to enhance oligodendrogenesis and CNS myelination in vivo.

Pathway-Specific Control of Striatal Neuron Vulnerability by Corticostriatal Cannabinoid CB1 Receptors.

The vast majority of neurons within the striatum are GABAergic medium spiny neurons (MSNs), which receive glutamatergic input from the cortex and thalamus, and form two major efferent pathways: the direct pathway, expressing dopamine D1 receptor (D1R-MSNs), and the indirect pathway, expressing dopamine D2 receptor (D2R-MSNs). While molecular mechanisms of MSN degeneration have been identified in animal models of striatal damage, the molecular factors that dictate a selective vulnerability of D1R-MSNs or D2R-MSNs remain unknown. Here, we combined genetic, chemogenetic, and pharmacological strategies with behavioral and neurochemical analyses, and show that the pool of cannabinoid CB1 receptor (CB1R) located on corticostriatal terminals efficiently safeguards D1R-MSNs, but not D2R-MSNs, from different insults. This cell-specific response relies on the regulation of glutamatergic signaling, and is independent from the CB1R-dependent control of astroglial activity in the striatum. These findings define cortical CB1R as a pivotal synaptic player in dictating a differential vulnerability of D1R-MSNs versus D2R-MSNs, and increase our understanding of the role of coordinated cannabinergic-glutamatergic signaling in establishing corticostriatal circuits and its dysregulation in neurodegenerative diseases.

Loss of Cannabinoid CB1 Receptors Induces Cortical Migration Malformations and Increases Seizure Susceptibility.

Neuronal migration is a fundamental process of brain development, and its disruption underlies devastating neurodevelopmental disorders. The transcriptional programs governing this process are relatively well characterized. However, how environmental cues instruct neuronal migration remains poorly understood. Here, we demonstrate that the cannabinoid CB1 receptor is strictly required for appropriate pyramidal neuron migration in the developing cortex. Acute silencing of the CB1 receptor alters neuronal morphology and impairs radial migration. Consequently, CB1 siRNA-electroporated mice display cortical malformations mimicking subcortical band heterotopias and increased seizure susceptibility in adulthood. Importantly, rescuing the CB1 deficiency-induced radial migration arrest by knockdown of the GTPase protein RhoA restored the hyperexcitable neuronal network and seizure susceptibility. Our findings show that CB1 receptor/RhoA signaling regulates pyramidal neuron migration, and that deficient CB1 receptor signaling may contribute to cortical development malformations leading to refractory epilepsy independently of its canonical neuromodulatory role in the adult brain.

Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons.

The CB1 cannabinoid receptor, the main target of Δ(9)-tetrahydrocannabinol (THC), the most prominent psychoactive compound of marijuana, plays a crucial regulatory role in brain development as evidenced by the neurodevelopmental consequences of its manipulation in animal models. Likewise, recreational cannabis use during pregnancy affects brain structure and function of the progeny. However, the precise neurobiological substrates underlying the consequences of prenatal THC exposure remain unknown. As CB1 signaling is known to modulate long-range corticofugal connectivity, we analyzed the impact of THC exposure on cortical projection neuron development. THC administration to pregnant mice in a restricted time window interfered with subcerebral projection neuron generation, thereby altering corticospinal connectivity, and produced long-lasting alterations in the fine motor performance of the adult offspring. Consequences of THC exposure were reminiscent of those elicited by CB1 receptor genetic ablation, and CB1-null mice were resistant to THC-induced alterations. The identity of embryonic THC neuronal targets was determined by a Cre-mediated, lineage-specific, CB1 expression-rescue strategy in a CB1-null background. Early and selective CB1 reexpression in dorsal telencephalic glutamatergic neurons but not forebrain GABAergic neurons rescued the deficits in corticospinal motor neuron development of CB1-null mice and restored susceptibility to THC-induced motor alterations. In addition, THC administration induced an increase in seizure susceptibility that was mediated by its interference with CB1-dependent regulation of both glutamatergic and GABAergic neuron development. These findings demonstrate that prenatal exposure to THC has long-lasting deleterious consequences in the adult offspring solely mediated by its ability to disrupt the neurodevelopmental role of CB1 signaling.

Sustained Gq-Protein Signaling Disrupts Striatal Circuits via JNK.

The dorsal striatum is a major input structure of the basal ganglia and plays a key role in the control of vital processes such as motor behavior, cognition, and motivation. The functionality of striatal neurons is tightly controlled by various metabotropic receptors. Whereas the Gs/Gi-protein-dependent tuning of striatal neurons is fairly well known, the precise impact and underlying mechanism of Gq-protein-dependent signals remain poorly understood. Here, using different experimental approaches, especially designer receptor exclusively activated by designer drug (DREADD) chemogenetic technology, we found that sustained activation of Gq-protein signaling impairs the functionality of striatal neurons and we unveil the precise molecular mechanism underlying this process: a phospholipase C/Ca2+/proline-rich tyrosine kinase 2/cJun N-terminal kinase pathway. Moreover, engagement of this intracellular signaling route was functionally active in the mouse dorsal striatum in vivo, as proven by the disruption of neuronal integrity and behavioral tasks. To analyze this effect anatomically, we manipulated Gq-protein-dependent signaling selectively in neurons belonging to the direct or indirect striatal pathway. Acute Gq-protein activation in direct-pathway or indirect-pathway neurons produced an enhancement or a decrease, respectively, of activity-dependent parameters. In contrast, sustained Gq-protein activation impaired the functionality of direct-pathway and indirect-pathway neurons and disrupted the behavioral performance and electroencephalography-related activity tasks controlled by either anatomical framework. Collectively, these findings define the molecular mechanism and functional relevance of Gq-protein-driven signals in striatal circuits under normal and overactivated states. SIGNIFICANCE STATEMENT: The dorsal striatum is a major input structure of the basal ganglia and plays a key role in the control of vital processes such as motor behavior, cognition, and motivation. Whereas the Gs/Gi-protein-dependent tuning of striatal neurons is fairly well known, the precise impact and underlying mechanism of Gq-protein-dependent signals remain unclear. Here, we show that striatal circuits can be "turned on" by acute Gq-protein signaling or "turned off" by sustained Gq-protein signaling. Specifically, sustained Gq-protein signaling inactivates striatal neurons by an intracellular pathway that relies on cJun N-terminal kinase. Overall, this study sheds new light onto the molecular mechanism and functional relevance of Gq-protein-driven signals in striatal circuits under normal and overactivated states.

Cannabinoid Type-2 Receptor Drives Neurogenesis and Improves Functional Outcome After Stroke.

BACKGROUND AND PURPOSE: Stroke is a leading cause of adult disability characterized by physical, cognitive, and emotional disturbances. Unfortunately, pharmacological options are scarce. The cannabinoid type-2 receptor (CB2R) is neuroprotective in acute experimental stroke by anti-inflammatory mechanisms. However, its role in chronic stroke is still unknown. METHODS: Stroke was induced by permanent middle cerebral artery occlusion in mice; CB2R modulation was assessed by administering the CB2R agonist JWH133 ((6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) or the CB2R antagonist SR144528 (N-[(1S)-endo-1,3,3-trimethylbicyclo-[2.2.1]-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) once daily from day 3 to the end of the experiment or by CB2R genetic deletion. Analysis of immunofluorescence-labeled brain sections, 5-bromo-2´-deoxyuridine (BrdU) staining, fluorescence-activated cell sorter analysis of brain cell suspensions, and behavioral tests were performed. RESULTS: SR144528 decreased neuroblast migration toward the boundary of the infarct area when compared with vehicle-treated mice 14 days after middle cerebral artery occlusion. Consistently, mice on this pharmacological treatment, like mice with CB2R genetic deletion, displayed a lower number of new neurons (NeuN+/BrdU+ cells) in peri-infarct cortex 28 days after stroke when compared with vehicle-treated group, an effect accompanied by a worse sensorimotor performance in behavioral tests. The CB2R agonist did not affect neurogenesis or outcome in vivo, but increased the migration of neural progenitor cells in vitro; the CB2R antagonist alone did not affect in vitro migration. CONCLUSIONS: Our data support that CB2R is fundamental for driving neuroblast migration and suggest that an endocannabinoid tone is required for poststroke neurogenesis by promoting neuroblast migration toward the injured brain tissue, increasing the number of new cortical neurons and, conceivably, enhancing motor functional recovery after stroke.

A restricted population of CB1 cannabinoid receptors with neuroprotective activity.

The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. Of note, CB1 receptors are expressed at the synapses of two opposing (i.e., GABAergic/inhibitory and glutamatergic/excitatory) neuronal populations, so the activation of one and/or another receptor population may conceivably evoke different effects. Despite the widely reported neuroprotective activity of the CB1 receptor in animal models, the precise pathophysiological relevance of those two CB1 receptor pools in neurodegenerative processes is unknown. Here, we first induced excitotoxic damage in the mouse brain by (i) administering quinolinic acid to conditional mutant animals lacking CB1 receptors selectively in GABAergic or glutamatergic neurons, and (ii) manipulating corticostriatal glutamatergic projections remotely with a designer receptor exclusively activated by designer drug pharmacogenetic approach. We next examined the alterations that occur in the R6/2 mouse, a well-established model of Huntington disease, upon (i) fully knocking out CB1 receptors, and (ii) deleting CB1 receptors selectively in corticostriatal glutamatergic or striatal GABAergic neurons. The data unequivocally identify the restricted population of CB1 receptors located on glutamatergic terminals as an indispensable player in the neuroprotective activity of (endo)cannabinoids, therefore suggesting that this precise receptor pool constitutes a promising target for neuroprotective therapeutic strategies.

CB1 Cannabinoid Receptor-Dependent Activation of mTORC1/Pax6 Signaling Drives Tbr2 Expression and Basal Progenitor Expansion in the Developing Mouse Cortex.

The CB1 cannabinoid receptor regulates cortical progenitor proliferation during embryonic development, but the molecular mechanism of this action remains unknown. Here, we report that CB1-deficient mouse embryos show premature cell cycle exit, decreased Pax6- and Tbr2-positive cell number, and reduced mammalian target of rapamycin complex 1 (mTORC1) activation in the ventricular and subventricular cortical zones. Pharmacological stimulation of the CB1 receptor in cortical slices and progenitor cell cultures activated the mTORC1 pathway and increased the number of Pax6- and Tbr2-expressing cells. Likewise, acute CB1 knockdown in utero reduced mTORC1 activation and cannabinoid-induced Tbr2-positive cell generation. Luciferase reporter and chromatin immunoprecipitation assays revealed that the CB1 receptor drives Tbr2 expression downstream of Pax6 induction in an mTORC1-dependent manner. Altogether, our results demonstrate that the CB1 receptor tunes dorsal telencephalic progenitor proliferation by sustaining the transcriptional activity of the Pax6-Tbr2 axis via the mTORC1 pathway, and suggest that alterations of CB1 receptor signaling, by producing the missexpression of progenitor identity determinants may contribute to neurodevelopmental alterations.

The CB(1) cannabinoid receptor drives corticospinal motor neuron differentiation through the Ctip2/Satb2 transcriptional regulation axis.

The generation and specification of pyramidal neuron subpopulations during development relies on a complex network of transcription factors. The CB(1) cannabinoid receptor is the major molecular target of endocannabinoids and marijuana active compounds. This receptor has been shown to influence neural progenitor proliferation and axonal growth, but its involvement in neuronal differentiation and the functional impact in the adulthood caused by altering its signaling during brain development are not known. Here we show that the CB(1) receptor, by preventing Satb2 (special AT-rich binding protein 2)-mediated repression, increased Ctip2 (COUP-TF interacting protein 2) promoter activity, and Ctip2-positive neuron generation. Unbalanced neurogenic fate determination found in complete CB(1)(-/-) mice and in glutamatergic neuron-specific Nex-CB(1)(-/-) mice induced overt alterations in corticospinal motor neuron generation and subcerebral connectivity, thereby resulting in an impairment of skilled motor function in adult mice. Likewise, genetic deletion of CB(1) receptors in Thy1-YFP-H mice elicited alterations in corticospinal tract development. Altogether, these data demonstrate that the CB(1) receptor contributes to the generation of deep-layer cortical neurons by coupling endocannabinoid signals from the neurogenic niche to the intrinsic proneurogenic Ctip2/Satb2 axis, thus influencing appropriate subcerebral projection neuron specification and corticospinal motor function in the adulthood.

CB2 cannabinoid receptors promote neural progenitor cell proliferation via mTORC1 signaling.

The endocannabinoid system is known to regulate neural progenitor (NP) cell proliferation and neurogenesis. In particular, CB(2) cannabinoid receptors have been shown to promote NP proliferation. As CB(2) receptors are not expressed in differentiated neurons, CB(2)-selective agonists are promising candidates to manipulate NP proliferation and indirectly neurogenesis by overcoming the undesired psychoactive effects of neuronal CB(1) cannabinoid receptor activation. Here, by using NP cells, brain organotypic cultures, and in vivo animal models, we investigated the signal transduction mechanism involved in CB(2) receptor-induced NP cell proliferation and neurogenesis. Exposure of hippocampal HiB5 NP cells to the CB(2) receptor-selective agonist HU-308 led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, which, by inhibiting its downstream target p27Kip1, induced NP proliferation. Experiments conducted with the CB(2) receptor-selective antagonist SR144528, inhibitors of the PI3K/Akt/mTORC1 axis, and CB(2) receptor transient-transfection vector further supported that CB(2) receptors control NP cell proliferation via activation of mTORC1 signaling. Likewise, CB(2) receptor engagement induced cell proliferation in an mTORC1-dependent manner both in embryonic cortical slices and in adult hippocampal NPs. Thus, HU-308 increased ribosomal protein S6 phosphorylation and 5-bromo-2'-deoxyuridine incorporation in wild-type but not CB(2) receptor-deficient NPs of the mouse subgranular zone. Moreover, adult hippocampal NP proliferation induced by HU-308 and excitotoxicity was blocked by the mTORC1 inhibitor rapamycin. Altogether, these findings provide a mechanism of action and a rationale for the use of nonpsychotomimetic CB(2) receptor-selective ligands as a novel strategy for the control of NP cell proliferation and neurogenesis.

The anxiolytic effect of cannabidiol on chronically stressed mice depends on hippocampal neurogenesis: involvement of the endocannabinoid system.

Cannabidiol (CBD), the main non-psychotomimetic component of the plant Cannabis sativa, exerts therapeutically promising effects on human mental health such as inhibition of psychosis, anxiety and depression. However, the mechanistic bases of CBD action are unclear. Here we investigate the potential involvement of hippocampal neurogenesis in the anxiolytic effect of CBD in mice subjected to 14 d chronic unpredictable stress (CUS). Repeated administration of CBD (30 mg/kg i.p., 2 h after each daily stressor) increased hippocampal progenitor proliferation and neurogenesis in wild-type mice. Ganciclovir administration to GFAP-thymidine kinase (GFAP-TK) transgenic mice, which express thymidine kinase in adult neural progenitor cells, abrogated CBD-induced hippocampal neurogenesis. CBD administration prevented the anxiogenic effect of CUS in wild type but not in GFAP-TK mice as evidenced in the novelty suppressed feeding test and the elevated plus maze. This anxiolytic effect of CBD involved the participation of the CB1 cannabinoid receptor, as CBD administration increased hippocampal anandamide levels and administration of the CB1-selective antagonist AM251 prevented CBD actions. Studies conducted with hippocampal progenitor cells in culture showed that CBD promotes progenitor proliferation and cell cycle progression and mimics the proliferative effect of CB1 and CB2 cannabinoid receptor activation. Moreover, antagonists of these two receptors or endocannabinoid depletion by fatty acid amide hydrolase overexpression prevented CBD-induced cell proliferation. These findings support that the anxiolytic effect of chronic CBD administration in stressed mice depends on its proneurogenic action in the adult hippocampus by facilitating endocannabinoid-mediated signalling.

Cannabinoids, Neurogenesis and Antidepressant Drugs: Is there a Link?

Similar to clinically used antidepressants, cannabinoids can also regulate anxiety and depressive symptoms. Although the mechanisms of these effects are not completely understood, recent evidence suggests that changes in endocannabinoid system could be involved in some actions of antidepressants. Chronic antidepressant treatment modifies the expression of CB1 receptors and endocannabinoid (EC) content in brain regions related to mood and anxiety control. Moreover, both antidepressant and cannabinoids activate mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase(PI3-K)/Akt or PKB signaling, intracellular pathways that regulate cell proliferation and neural cell survival. Facilitation of hippocampal neurogenesis is proposed as a common effect of chronic antidepressant treatment. Genetic or pharmacological manipulations of cannabinoid receptors (CB1 and CB2) or enzymes responsible for endocannabinoid-metabolism have also been shown to control proliferation and neurogenesis in the hippocampus. In the present paper we reviewed the studies that have investigated the potential contribution of cannabinoids and neurogenesisto antidepressant effects. Considering the widespread brain distribution of the EC system, a better understanding of this possible interaction could contribute to the development of therapeutic alternatives to mood and anxiety disorders.

Control of a hippocampal recurrent excitatory circuit by cannabinoid receptor-interacting protein Gap43.

The type-1 cannabinoid receptor (CB1R) is widely expressed in excitatory and inhibitory nerve terminals, and by suppressing neurotransmitter release, its activation modulates neural circuits and brain function. While the interaction of CB1R with various intracellular proteins is thought to alter receptor signaling, the identity and role of these proteins are poorly understood. Using a high-throughput proteomic analysis complemented with an array of in vitro and in vivo approaches in the mouse brain, we report that the C-terminal, intracellular domain of CB1R interacts specifically with growth-associated protein of 43 kDa (GAP43). The CB1R-GAP43 interaction occurs selectively at mossy cell axon boutons, which establish excitatory synapses with dentate granule cells in the hippocampus. This interaction impairs CB1R-mediated suppression of mossy cell to granule cell transmission, thereby inhibiting cannabinoid-mediated anti-convulsant activity in mice. Thus, GAP43 acts as a synapse type-specific regulatory partner of CB1R that hampers CB1R-mediated effects on hippocampal circuit function.

Loss of striatal type 1 cannabinoid receptors is a key pathogenic factor in Huntington's disease.

Endocannabinoids act as neuromodulatory and neuroprotective cues by engaging type 1 cannabinoid receptors. These receptors are highly abundant in the basal ganglia and play a pivotal role in the control of motor behaviour. An early downregulation of type 1 cannabinoid receptors has been documented in the basal ganglia of patients with Huntington's disease and animal models. However, the pathophysiological impact of this loss of receptors in Huntington's disease is as yet unknown. Here, we generated a double-mutant mouse model that expresses human mutant huntingtin exon 1 in a type 1 cannabinoid receptor-null background, and found that receptor deletion aggravates the symptoms, neuropathology and molecular pathology of the disease. Moreover, pharmacological administration of the cannabinoid Δ(9)-tetrahydrocannabinol to mice expressing human mutant huntingtin exon 1 exerted a therapeutic effect and ameliorated those parameters. Experiments conducted in striatal cells show that the mutant huntingtin-dependent downregulation of the receptors involves the control of the type 1 cannabinoid receptor gene promoter by repressor element 1 silencing transcription factor and sensitizes cells to excitotoxic damage. We also provide in vitro and in vivo evidence that supports type 1 cannabinoid receptor control of striatal brain-derived neurotrophic factor expression and the decrease in brain-derived neurotrophic factor levels concomitant with type 1 cannabinoid receptor loss, which may contribute significantly to striatal damage in Huntington's disease. Altogether, these results support the notion that downregulation of type 1 cannabinoid receptors is a key pathogenic event in Huntington's disease, and suggest that activation of these receptors in patients with Huntington's disease may attenuate disease progression.

Cannabinoid CB1 receptor gene inactivation in oligodendrocyte precursors disrupts oligodendrogenesis and myelination in mice.

Cannabinoids are known to modulate oligodendrogenesis and developmental CNS myelination. However, the cell-autonomous action of these compounds on oligodendroglial cells in vivo, and the molecular mechanisms underlying these effects have not yet been studied. Here, by using oligodendroglial precursor cell (OPC)-targeted genetic mouse models, we show that cannabinoid CB1 receptors exert an essential role in modulating OPC differentiation at the critical periods of postnatal myelination. We found that selective genetic inactivation of CB1 receptors in OPCs in vivo perturbs oligodendrogenesis and postnatal myelination by altering the RhoA/ROCK signaling pathway, leading to hypomyelination, and motor and cognitive alterations in young adult mice. Conversely, pharmacological CB1 receptor activation, by inducing E3 ubiquitin ligase-dependent RhoA proteasomal degradation, promotes oligodendrocyte development and CNS myelination in OPCs, an effect that was not evident in OPC-specific CB1 receptor-deficient mice. Moreover, pharmacological inactivation of ROCK in vivo overcomes the defects in oligodendrogenesis and CNS myelination, and behavioral alterations found in OPC-specific CB1 receptor-deficient mice. Overall, this study supports a cell-autonomous role for CB1 receptors in modulating oligodendrogenesis in vivo, which may have a profound impact on the scientific knowledge and therapeutic manipulation of CNS myelination by cannabinoids.

Endocannabinoid signaling controls pyramidal cell specification and long-range axon patterning.

Endocannabinoids (eCBs) have recently been identified as axon guidance cues shaping the connectivity of local GABAergic interneurons in the developing cerebrum. However, eCB functions during pyramidal cell specification and establishment of long-range axonal connections are unknown. Here, we show that eCB signaling is operational in subcortical proliferative zones from embryonic day 12 in the mouse telencephalon and controls the proliferation of pyramidal cell progenitors and radial migration of immature pyramidal cells. When layer patterning is accomplished, developing pyramidal cells rely on eCB signaling to initiate the elongation and fasciculation of their long-range axons. Accordingly, CB(1) cannabinoid receptor (CB(1)R) null and pyramidal cell-specific conditional mutant (CB(1)R(f/f,NEX-Cre)) mice develop deficits in neuronal progenitor proliferation and axon fasciculation. Likewise, axonal pathfinding becomes impaired after in utero pharmacological blockade of CB(1)Rs. Overall, eCBs are fundamental developmental cues controlling pyramidal cell development during corticogenesis.

Δ9 -Tetrahydrocannabinol promotes functional remyelination in the mouse brain.

BACKGROUND AND PURPOSE: Research on demyelinating disorders aims to find novel molecules that are able to induce oligodendrocyte precursor cell differentiation to promote central nervous system remyelination and functional recovery. Δ9 -Tetrahydrocannabinol (THC), the most prominent active constituent of the hemp plant Cannabis sativa, confers neuroprotection in animal models of demyelination. However, the possible effect of THC on myelin repair has never been studied. EXPERIMENTAL APPROACH: By using oligodendroglia-specific reporter mouse lines in combination with two models of toxin-induced demyelination, we analysed the effect of THC on the processes of oligodendrocyte regeneration and functional remyelination. KEY RESULTS: We show that THC administration enhanced oligodendrocyte regeneration, white matter remyelination and motor function recovery. THC also promoted axonal remyelination in organotypic cerebellar cultures. THC remyelinating action relied on the induction of oligodendrocyte precursor differentiation upon cell cycle exit and via CB1 cannabinoid receptor activation. CONCLUSIONS AND IMPLICATIONS: Overall, our study identifies THC administration as a promising pharmacological strategy aimed to promote functional CNS remyelination in demyelinating disorders.

Microglial CB2 cannabinoid receptors are neuroprotective in Huntington's disease excitotoxicity.

Cannabinoid-derived drugs are promising agents for the development of novel neuroprotective strategies. Activation of neuronal CB(1) cannabinoid receptors attenuates excitotoxic glutamatergic neurotransmission, triggers prosurvival signalling pathways and palliates motor symptoms in animal models of neurodegenerative disorders. However, in Huntington's disease there is a very early downregulation of CB(1) receptors in striatal neurons that, together with the undesirable psychoactive effects triggered by CB(1) receptor activation, foster the search for alternative pharmacological treatments. Here, we show that CB(2) cannabinoid receptor expression increases in striatal microglia of Huntington's disease transgenic mouse models and patients. Genetic ablation of CB(2) receptors in R6/2 mice, that express human mutant huntingtin exon 1, enhanced microglial activation, aggravated disease symptomatology and reduced mice lifespan. Likewise, induction of striatal excitotoxicity in CB(2) receptor-deficient mice by quinolinic acid administration exacerbated brain oedema, microglial activation, proinflammatory-mediator state and medium-sized spiny neuron degeneration. Moreover, administration of CB(2) receptor-selective agonists to wild-type mice subjected to excitotoxicity reduced neuroinflammation, brain oedema, striatal neuronal loss and motor symptoms. Studies on ganciclovir-induced depletion of astroglial proliferation in transgenic mice expressing thymidine kinase under the control of the glial fibrillary acidic protein promoter excluded the participation of proliferating astroglia in CB(2) receptor-mediated actions. These findings support a pivotal role for CB(2) receptors in attenuating microglial activation and preventing neurodegeneration that may pave the way to new therapeutic strategies for neuroprotection in Huntington's disease as well as in other neurodegenerative disorders with a significant excitotoxic component.