Non-psychotropic cannabinoids are receiving increasing attention due to their biological properties.

In this study, the NSC-34 motor neuron-like cells were treated with CBG (1 or 5 µM) or CBD (1 or 5 µM). MTT assay showed that both CBG and CBD at the tested doses were not cytotoxic, as confirmed by transcriptomic analysis that evidenced the downregulation of genes involved in cell death mechanisms such as MAP3K5, PARP4, EIRF2AK3, ERN1, CASP6 and CASP8 (Table 1). On the contrary, the data indicated that both compounds upregulated pro-survival genes, such as AKT1, ATF4 and BCL2L1. In our analysis, treatment with CBG and CBD, especially at the concentration 5 µM, induced the upregulation of this gene. Moreover, CBG downregulated CASP8 at low dose; while CBD downregulate CASP6 and CASP8 at 5 µM concentration. These results indicated that CBD at 5 µM is more efficacious in downregulating caspases. Regarding the pro-survival genes, AKT1 encodes the serine-threonine protein kinase 1 (Akt1), belonging to Akt family, that promotes cellular survival, phosphorylating and inhibiting death-inducing proteins. Akt1 also regulates cell survival via the phosphorylation of MAP3K5, a kinase related to the apoptotic signal [19]. Therefore, Akt1-mediated phosphorylation of MAP3K5 reduces its activity and consequently prevents apoptosis. In compliance with this evidence, our results showed that CBG and CBD, at both doses, reduced the expression of MAP3K5. BCL2L1 encodes a pro-survival protein belonging to the Bcl-2 family.

In this study, using NGS technology, we wanted only to analyze the transcriptomic profile of NSC-34 motor neuron-like cells treated with CBG or CBD at doses of 1 or 5 µM, in order to compare the effects of these two phytocompounds and evaluate how they modified the transcriptomic profile of the principal neurotransmission pathways. Our transcriptomic analysis demonstrated that treatment of NSC-34 motor neuron-like cells with CBG and CBD influenced several genes related to signaling pathways of the nervous system such as “Dopaminergic synapse”, “Glutamatergic Synapse” and “GABAergic synapse”. Specifically, CBG treatment modulates the same genes as CBD, inducing a similar transcriptomic profile.

Our results showed that in the “Dopaminergic synapse”, treatment with CBG and CBD downregulated the expression of SLC18A, DRD2, GNAI, PRKCB, CAMK2, CREB1 and CLOCK. Conversely, both phytocompounds promoted the expression of seven genes (DRD4, ADCY5, AKT1, ATF4, GNAI2, GNB4 and PRKCA). CBG downregulated the SLC18A1 gene, more at the dose of 5 µM; while CBD strongly downregulated this gene at 1 µM dose. SLC18A1 encodes for vesicular monoamine transporter 2, the principal of monoaminergic neurons. This transporter is present in the pre-synaptic vesicles of all monoaminergic neurons, and it is the common mechanism for the energy-driven uptake of dopamine [20]. It is worth noting that CBG downregulated the DRD2 gene in a dose independent manner, while CBD downregulated this gene more at the highest dose. Conversely, both phytocompounds upregulated the DRD4 gene, especially at the 5 µM dose. These genes encode Dopamine Receptor D2 and Dopamine Receptor D4, respectively. These receptors belong to the D2-like family and are associated with inhibiting G proteins (Gα_i/o); therefore, they determine a reduction in cyclic adenosine monophosphate (cAMP) which causes the opening of potassium channels and closing of those of calcium. Furthermore, in our NSC-34 cells, CBG and CBD, in a dose-independent manner, downregulated the CREB1 gene, which encodes the cAMP-response element-binding protein (CREB). However, our transcriptional analysis showed that both phytocompounds upregulated ATF4 gene, which also encodes for CREB2. CREB is located in the nucleus and is a transcription factor, which binds to the cAMP response element (CRE) of the promoters of its target genes. The protein is phosphorylated by various protein kinases such as mitogen-activated protein kinases (MAPK) and calmodulin-dependent protein kinase (CaMK) [21]. In our NSC-34 motor neuron-like cells, treatment with both phytocompounds reduced the expression of the CAMK2B gene, which encodes CaMK II Beta. CREB is involved in numerous intracellular processes, including proliferation, differentiation, survival, long-term synaptic enhancement, neurogenesis and neuronal plasticity [22]. Therefore, compounds such as CBG and CBD, by modulating the expression of this gene, could balance the phosphorylation levels of CREB.

Glutamate and GABA are two amino acid neurotransmitters that play an important role in controlling neuronal excitability [23]. To our knowledge, there are limited data about CBG influence on synaptic transmission. The CBG quinone derivative VCE-003.2 was reported to be able to reduce glial glutamate transporters [24]. On the contrary, CBD is known to be involved in the regulation of the excitatory transmission of glutamate and the inhibitory transmission of GABA [25]. In particular, CBD appears to be a GABA_A receptor modulator, being able to increase GABA-evoked currents [26].

Our results demonstrated that CBG and CBD upregulated the GNAI2 gene. This gene encodes for the Guanine nucleotide-binding protein G (i) subunit alpha (α)-2. The guanine nucleotide-binding proteins are made of Gα, beta (β) and gamma (γ) subunits [27]. The α chain contains the guanine nucleotide-binding site and shows a GTPase activity that converts associated GTP to GDP, thus interrupting the signal. The Gβ and Gγ subunits form stable functional complexes that can regulate numerous effectors such as ion channels, enzymes and various kinases [28]. The β subunit is encoded by five genes (GNB1-5). In our transcriptomic analysis, CBD positively regulated GNB4 in a dose-dependent manner. Conversely, the higher the dose of CBG, the less the gene is upregulated. Results from previous studies indicated that Gβ4 is widely expressed, can form dimers with nearly all Gγ subunits and modulated calcium, potassium channels and phospholipase isoforms [29]. β subunits of G proteins are associated with γ subunits, in order to form a Gβγ dimers, that affected glutamate transporter 1 (GLT-1) activity. In particular, GNB4 modulates the activity of glutamate transporter, suggesting a physical and functional interaction between GLT-1 and Gβγ dimers [30]. It has been shown that the subunits β2 are localized in the postsynaptic compartment, suggesting that Gβ-mediated signaling may be important for synapse formation and/or function [31].

The Gα_i/o, including the subunit encoded by GNAI2, negatively couples with adenylyl cyclase (AC) with a consequent reduction in cAMP levels. A decrease in cAMP levels reduces protein kinase A (PKA) activity, reducing the release of neurotransmitters [32]. Our bioinformatic analysis, in association and according to the upregulation of GNAI2, reported that CBG and CBD downregulated, in a dose-dependent manner, the ADCY9. The ADCY9 (adenylate cyclase 9) is the main gene involved in the signaling of the second messenger. Then, all together, these results may indicate that CBG treatment in NSC-34 motor neuron-like cells, and similarly CBD, may reduce the release of neurotransmitters through the G_i/o-mediated inhibition of cAMP/PKA. Conversely, CBG increased the expression of ADCY5, more at the 1 µM dose compared to the 5 µM; while CBD upregulated ADCY5 in a dose-dependent manner. ADCY5 encodes for adenylate cyclase 5. The activity of ADCY5 in motor neurons or in the peripheral nervous system remains to be clarified.

Our results showed that CBG and CBD also regulated genes involved in glutamate reuptake. After release from the presynaptic terminals, glutamate is quickly removed from the synaptic clefts by a family of five glutamate transporters, knows as excitatory amino acid transporters (EAAT1–5). EAATs are dual-function transport proteins: they are glutamate transporters and also act as anion channels [33]. CBG and CBD, at all doses, decreased the expression of the SLC1A2 that encodes for EAAT2. The EAAT2 transporters are responsible for 90% of glutamate reuptake into the nervous system [34]. For these reasons, the modulation of EAATs in motor neurons by CBG and CBD is very interesting and allows the knowledge about phytocompound effects to be deepened.

Moreover, CBG downregulated, in a dose-dependent manner, the SLC38A1 gene, downregulating it more at lower doses. Similarly, CBD modulates this gene. This gene encodes the glutamine transporter (GlnT) expressed in the membranes of glutamatergic neurons [35]. However, it has been shown that SLC38A1 is also expressed in GABAergic neurons; therefore, it could be involved in the supply of the GABA neurotransmitter [36]. In this way, CBG and CBD might interact with the excitatory glutamatergic transmission.

In the postsynaptic compartment, CBG and CBD, at both doses, increased the expression of DLGAP1 that encoded for Discs large associated proteins 1 (DLGAPs), also known as GKAP or SAPAP1. DLGAPs proteins that play an important role in the postsynaptic density are scaffold proteins and are involved in signaling glutamate receptors [37]. The DLGAP family may also have a role in modulating the turnover of ionotropic and metabotropic glutamate receptors in response to synaptic activity [38]. Therefore, DLGAPs are relevant for maintaining excitatory synapses. DLGAP proteins bind a second family of proteins, multiple ankyrin repeat domain proteins (SHANK) [39]. The SHANK family is characterized by a domain organization consisting of ankyrin repeats near the N terminal, followed by the SH3 domain, postsynaptic density protein-95, disk-large tumor suppressor protein, zonula occludens-1 (PDZ) domain, proline-rich region and a sterile alpha motif (SAM) domain at the C end. This organization allows them to mediate multiple interactions [40]. In our NSC-34 motor neuron-like cells, CBG downregulated the SHANK1 gene more at the lower dose. Conversely, CBD downregulated this gene in a dose-dependent manner. SHANK proteins are expressed in the nervous system and are more concentrated in the excitatory synapses [40,41,42]. SHANK proteins are scaffold proteins that connect neurotransmitter receptors with other membrane proteins, with cytoskeleton or other protein to mediate the signal. These proteins mediate a platform needed for G-protein-mediated signaling. SHANK proteins are also involved in morphological changes, being involved in the maturation of dendritic spines and formation of the synapses [43]. These proteins play a key role in neurotransmission due to their ability to link metabotropic glutamate receptors (mGluRs) to ionotropic glutamate receptors, N-methyl-D-aspartate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl- 4-isoxazole propionic acid receptors (AMPARs) [44,45]. Indeed, the PDZ domain of SHANK binds to the DLGAP family of proteins that in turn bind PSD-95, leading to the link of SHANK to the NMDA receptor [39]. Furthermore, SHANK binds also Homer which binds to group 1 and metabotropic glutamate receptors. Therefore, SHANK, due to its interactions with DLGAP and Homer, has the ability to link together the NMDA receptor complexes and the metabotropic glutamate receptor in the excitatory postsynaptic terminal [46]. DLGAP and SHANK are mutually dependent on each other, and these complexes play a key role in the correct positioning of many components in the excitatory postsynaptic terminal and the interactions between them [39]. For these reasons, the modulation of the DLGAP/SHANK pathway in NSC-34 motor neuron-like cells by CBG or CBD may alter the activity of the excitatory synapse and reduce the excitatory synaptic transmission. Indeed, it has been shown that the reduction in SHANK causes alterations of the DLGAP, NMDAR and AMPAR levels [47,48] with a consequent reduction in the number of synapses [46,49].

Interestingly, our results showed that CBG, and similarly CBD, negatively regulated the expression of the PPP3R1 gene. However, conversely to CBD, CBG downregulated this gene more at the lower dose. The PPP3R1 gene encodes for calcineurin, a serine/threonine phosphatase activated by calcium and calmodulin. This protein promotes the dephosphorylation of numerous biological targets, such as pro-apoptotic factors, transcription factors and ion channels [50]. Furthermore, it has been shown that it plays a key role in the apoptosis processes of neuronal cells, induced by neuroexcitotoxic stimuli due to the excessive release of glutamate [51]. In our analysis, CBG and CBD downregulated the PRKCB and PLD1 genes encoding for protein kinase C (PKC) β and phospholipase D1 (PLD1), respectively. On the contrary, they slightly upregulated the PRKCA gene that encodes for PKCα. These proteins are involved in the downstream processes of the selective activation of mGluRs. Therefore, the strong downregulation of PRKCB and PLD1 could reduce the postsynaptic glutamatergic activity. A novelty of this study is the finding that CBG can exert actions similar to CBD, reducing glutamate signaling in NSC-34 motor neuron-like cells.

Interestingly, our data also evidenced that CBG and similarly CBD influenced the gene expression involved in GABAergic transmission—specifically, CBG and CBD downregulation of the SLC38A1 gene while they upregulated the SLC32A1 gene. This gene encodes for solute carrier family 32 member 1, also known as vesicular GABA transporter, involved in the uptake of GABA into the synaptic vesicles. Our results demonstrate that CBG and CBD upregulating SLC32A1 could potentiate inhibitory postsynaptic currents.

Our analysis demonstrated that NSC-34 motor neuron-like cells treated with CBG and with CBD showed the upregulation of HAP1 gene, encoding Huntingtin-associated protein-1 (Hap1). This protein binds directly to the GABA A receptors, preventing their degradation on the plasma membrane with consequent improvement of inhibitory synaptic transmission [52]. Overexpression of Hap1 in cultured neurons has been shown to increase the number of GABA A receptors on the surface of the cell membrane, thereby increasing the average amplitude of the currents evoked from GABA [53]. In accordance with this evidence, our results may suggest that CBG and CBD, overexpressing the HAP1, may be able to reduce neuronal excitability in our motor neuron-like cells by enhancing postsynaptic GABAergic activity. However, these data should be confirmed in synaptosomal preparation and differentiated neuronal cells.

Furthermore, CBG and CBD can also regulate the phosphorylation of the GABA_A receptor through PKC. In line with our results, several studies have shown that different PKC isoforms can regulate the function of the GABA A receptor [54]. In this article, we have shown that CBD reduced the expression of genes encoding glutamine transporters. Conversely, CBD enhances GABA_A receptor activity. The main result of this work is that in our transcriptomic analysis CBG exhibited similar actions to CBD both for dopaminergic, GABAergic and glutamatergic signaling. CBG induced similar transcriptional changes in genes involved in dopamine, GABA and glutamate pathways compared to CBD.