Changes in Endocannabinoid Receptors and Enzymes in the Spinal Cord of SOD1^G93A Transgenic Mice and Evaluation of a Sativex^®‐like Combination of Phytocannabinoids: Interest for Future Therapies in Amyotrophic Lateral Sclerosis

Summary

Aims

Cannabinoids afford neuroprotection in SOD1^G93A mutant mice, an experimental model of amyotrophic lateral sclerosis (ALS). However, these mice have been poorly studied to identify alterations in those elements of the endocannabinoid system targeted by these treatments. Moreover, we studied the neuroprotective effect of the phytocannabinoid‐based medicine Sativex^® in these mice.

Methods

First, we analyzed the endocannabinoid receptors and enzymes in the spinal cord of SOD1^G93A transgenic mice at a late stage of the disease. Second, 10‐week‐old transgenic mice were daily treated with an equimolecular combination of Δ⁹‐tetrahydrocannabinol‐ and cannabidiol‐enriched botanical extracts (20 mg/kg for each phytocannabinoid).

Results

We found a significant increase of CB ₂ receptors and NAPE‐PLD enzyme in SOD1^G93A transgenic males and only CB ₂ receptors in females. Pharmacological experiments demonstrated that the treatment of these mice with the Sativex^®‐like combination of phytocannabinoids only produced weak improvements in the progression of neurological deficits and in the animal survival, particularly in females.

Conclusions

Our results demonstrated changes in endocannabinoid signaling, in particular a marked up‐regulation of CB ₂ receptors, in SOD1^G93A transgenic mice, and provide support that Sativex^® may serve as a novel disease‐modifying therapy in ALS.

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by the selective damage of motor neurons in the spinal cord, brainstem, and motor cortex 1. ALS exists in two forms, familial ALS (only 5% of cases) and sporadic ALS (most of cases) 1, 2. The pathogenesis of ALS is still pending of complete identification, but some mechanisms have been found to be involved including excitotoxic damage, chronic inflammation, oxidative stress, and protein aggregation 2, 3, 4. For example, several studies have identified changes in the function of glutamate transporters that have been associated with the initial phases of the disease 4. High amounts of reactive microglia have been found in those brain regions that are affected in ALS patients 5. Genetic studies have identified several mutations in the copper–zinc superoxide dismutase gene (SOD1), which encodes for a key antioxidant enzyme, in approximately 12% cases of familial ALS 6, being pathological through a gain‐of‐neurotoxic function. In the last years, mutations in other genes, such as TARDBP (TAR‐DNA binding protein) and FUS (fused in sarcoma), which encode proteins involved in pre‐mRNA splicing, transport and/or stability 7, 8, and, in particular, the CCGGGG hexanucleotide expansion in the C9orf72 gene, which appears to account for up to 40% of genetic cases 9, have been also identified and related to the disease. Their pathogenic mechanisms, which differ, in part, from the toxicity associated with mutations in SOD1, led to a novel molecular classification of ALS subtypes 10.

Despite the intensive investigation developed in the last years, the disease still lacks of an effective treatment, with Rilutek^® as the only approved medicine 11. Recent studies support that cannabinoids may be beneficial as neuroprotective agents in ALS 12. Thus, the motor impairment was delayed, and the animal survival prolonged after the treatment with the phytocannabinoid Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC) in the SOD1^G93A transgenic mouse model of ALS 13. Other cannabinoids, including the less psychotropic plant‐derived cannabinoid cannabinol 14, the nonselective agonist WIN55,212‐2 15, and the selective CB₂ agonist AM1241 16, 17, produced similar effects. The efficacy shown by compounds that target the CB₂ receptor 16, 17 correlates with the fact that this receptor is overexpressed in microglial cells in postmortem spinal cords from ALS patients 18 or SOD1^G93A transgenic mice 17, becoming a promising target for the development of disease‐modifying therapies in ALS, as has been investigated in other disorders 19, 20. This has been also proposed for the fatty acid amide hydrolase (FAAH) enzyme, which plays a key role in the degradation of the endocannabinoids anandamide and, to a lesser extent, 2‐arachidonoyl‐glycerol. Thus, elevated levels of endocannabinoids, reached by means of genetic ablation or pharmacological inhibition of this enzyme, also caused a delay in the appearance of the disease in SOD1^G93A transgenic mice 15. This was not seen with the genetic ablation of the CB₁ receptor, although these animals showed an increased survival rate 15. As for the CB₂ receptor, the efficacy of FAAH inhibition/inactivation 15 agrees with the elevated levels of anandamide found in the spinal cord of SOD1^G93A mutant mice 21, thus indicating that such response, as in the case of the CB₂ receptor, seems to represent an endogenous protective response against the insults that damage the motor neurons in the spinal cord, which would deserve to be pharmacologically exploited.

Despite the promising evidence supporting that cannabinoids may serve to develop a disease‐modifying therapy in ALS, several issues remain to be investigated. For example, it is poorly known how the disease affects other elements of the endocannabinoid signaling system, such as the CB₁ receptor, the key enzyme in anandamide synthesis, N‐arachidonoyl‐phosphatidylethanolamine‐phospholipase D (NAPE‐PLD) or its equivalent in 2‐arachidonoylglycerol synthesis, diacylglycerol lipase (DAGL), and the key enzyme in the degradation of 2‐arachidonoylglycerol, monoacylglycerol lipase (MAGL). We believe extremely important to determine the changes in these elements, as well as in FAAH enzyme and CB₂ receptor, in ALS, as these changes may greatly influence the efficacy of those therapies based on targeting the different elements of this signaling system. Therefore, the first objective of our study was to analyze the status of endocannabinoid receptors (e.g., CB₁ and CB₂) and enzymes (e.g., NAPE‐PLD, DAGL, FAAH, and MAGL), using RT‐PCR, in the spinal cord of SOD1^G93A transgenic mice at a late stage of the disease.

The pharmacological studies conducted so far in experimental ALS appear to indicate that neuroprotective properties of cannabinoids in ALS depend on the combination of different mechanisms. The data obtained in in vivo studies using the nonselective cannabinoid receptor agonist WIN55,212‐2 15 or compounds that selectively target the CB₂ receptor 16, 17 suggest the participation of cannabinoid receptors and, in particular, the CB₂ receptor type associated with the inflammatory role of glial elements in this disease. However, other mechanisms, such as those involved in cannabinoid receptor‐independent antioxidant properties of certain cannabinoids, for example, phytocannabinoids, cannot be excluded. The fact that the neuroprotective effects in experimental models of ALS were reached through the activation of multiple targets and the treatment with different cannabinoid compounds suggests the convenience of using a cannabinoid with a broad‐spectrum action or, alternatively, a combination of different cannabinoids with different profiles. This may be the case of Sativex^® (GW Pharmaceuticals Ltd, Cambridgeshire, UK), a cannabinoid‐based medicine that has been recently approved for the treatment of spasticity and pain in multiple sclerosis patients 22. Sativex^® combines botanical extracts enriched with Δ⁹‐THC and cannabidiol (CBD), which facilitates the activation of different mechanisms/targets, for example, both phytocannabinoids may act through cannabinoid receptor‐independent antioxidant mechanisms, whereas Δ⁹‐THC may activate CB₁ and CB₂ receptors 23. It is important to remark that the fact that Sativex^® is already licensed may facilitate the development of clinical studies in ALS patients in the case of positive effects. Therefore, the second objective of our study was to evaluate a Sativex^®‐like combination of phytocannabinoid botanical extracts (administered i.p. vs. the oromucosal form used in patients, which implies some differences in pharmacokinetics), as a disease‐modifying therapy in this experimental ALS model.

Materials and methods

Animals, Treatments and Sampling

All experiments were conducted with B6SJL‐Tg(SOD1*G93A)1Gur/J transgenic, and nontransgenic littermate sibling mice bred in our animal facilities from initial breeders generously provided by LagenBio‐Ingen (University of Zaragoza, Zaragoza, Spain) and subjected to genotyping for identifying the presence or absence of the transgene containing the SOD1^G93A mutation (protocol provided by LagenBio‐Ingen). All animals were housed in a room with controlled photoperiod (08:00–20:00 light) and temperature (22 ± 1°C) with free access to standard food and water. All experiments were conducted according to local and European rules (directive 2010/63/EU) and approved by the Committee for Animal Experimentation of our university. In a first experiment, we used nontransgenic and B6SJL‐Tg(SOD1*G93A)1Gur/J mutant mice of both genders at the age of 120 days after birth, an age at which, according to previous studies 21, 24, motor deficits in mutant mice are already evident and strongly disabling. Before being euthanized, animals were subjected to neurological examination to confirm the existence of such motor deficits according to the following scale: 5 = no symptoms; 4 = tremor in hindlimbs when suspended by the tail; 3 = gait anomalies; 2 = paralysis in one hindlimb; 1 = paralysis in both hindlimbs; and 0 = unability to turn when lying on the back for 15 seconds (see details in refs. 21 and 24). Immediately after, animals were euthanized and their spinal cords were rapidly removed, frozen in 2‐methylbutane cooled in dry ice, and stored at −80°C for subsequent biochemical analyses (qRT‐PCR). In this experiment, at least 6–8 animals were used per experimental group. In a second experiment, we conducted pharmacological studies with B6SJL‐Tg(SOD1*G93A)1Gur/J mutant mice and their nontransgenic littermate siblings starting the treatment at ages (9 weeks after birth in the case of males and 10 weeks in the case of females, as the disease initiates earliest in males than females; see Figure 4) at which, according to previous studies 21, 24, SOD1^G93A mutant mice show the first evidence of motor anomalies. Treatments consisted of a daily i.p. injection of a 1:1 combination of botanical extracts enriched with either Δ⁹‐THC (Δ⁹‐THC botanical extract contains 67.1% Δ⁹‐THC, 0.3% CBD, 0.9% cannabigerol, 0.9% cannabichromene, and 1.9% other phytocannabinoids) or CBD (CBD botanical extract contains 64.8% CBD, 2.3% Δ⁹‐THC, 1.1% cannabigerol, 3.0% cannabichromene, and 1.5% other phytocannabinoids), both provided by GW Pharmaceuticals Ltd. (Cambridgeshire, UK), or vehicle (Tween 80‐saline; 1:16). The total dose of cannabinoid administered was always 40 mg/kg (equivalent to 20 mg/kg of pure CBD + 20 mg/kg of pure Δ⁹‐THC), a dose within the range of effective doses of phytocannabinoids when they were administered in pure form in experimental models of neurodegenerative disorders including ALS 13, 14. The treatment was repeated every day up to the end stage of the disease, when animals reached a clinical score of 0 (they were euthanized to avoid animal suffering) enabling to evaluate animal survival using Kaplan–Meier curves. Every week, all animals were subjected to neurological examination following the clinical scale described before. In this experiment, at least 8–12 animals were used per experimental group.

Figure 4.

Clinical score and animal survival measured in male and female SOD‐1 transgenic mice daily treated, from the age of 9 (in males) or 10 (in females) weeks after birth, with the Sativex^®‐like combination of phytocannabinoids at a dose of 40 mg/kg (equivalent to 20 mg/kg for each major phytocannabinoid) or vehicle (Tween 80‐saline). Details in the text. Values are expressed as means ± SEM for 8–12 animals per group. Data were analyzed using two‐way analysis of variance followed by the Student–Newman–Keuls test (*P < 0.05 compared to animals treated with vehicle).

Real‐Time qRT‐PCR Analysis

Total RNA was extracted from spinal cord samples using Trizol (Life Technologies, Alcobendas, Spain) and purified using PureLink^® RNA Mini Kit RNATidy reagent (Life Technologies, Alcobendas, Spain). The total amount of RNA extracted was quantitated by spectrometry at 260 nm, and its purity was evaluated by the ratio between the absorbance values at 260 and 280 nm, whereas its integrity was confirmed in agarose gels. To prevent genomic DNA contamination, DNA was removed and single‐stranded complementary DNA was synthesized from 1 μg of total RNA using a commercial kit (Rneasy Mini Quantitect Reverse Transcription, Qiagen, Izasa, Madrid, Spain). The reaction mixture was kept frozen at −20°C until enzymatic amplification. Quantitative real‐time PCR assays were performed using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) to quantify mRNA levels for CB₁ receptor (ref. Mm00432621_s1), CB₂ receptor (ref. Mm00438286_m1), FAAH (ref. Mm00515684_m1), MAGL (ref. Mm00449274_m1), DAGL (ref. Mm00813830_m1), and NAPE‐PLD (ref. Mm00724596_m1) using GAPDH expression (ref. Mm99999915_g1) as an endogenous control gene for normalization. The PCR assay was performed using the 7300 Fast Real‐Time PCR System (Applied Biosystems), and the threshold cycle (Ct) was calculated by the instrument's software (7300 Fast System; Applied Biosystems).

Statistics

Data were assessed by unpaired Student's t‐test or two‐way ANOVA followed by the Student–Newman–Keuls test, as required.

Results

Experiment I: Analysis of the Endocannabinoid Signaling in SOD1^G93A Mutant Mice

In the first objective of this study, the spinal cord of 17‐week‐old animals was used for biochemical analysis of endocannabinoid receptors and enzymes using RT‐PCR. Our analyses proved a significant increase of CB₂ receptor expression in SOD1^G93A transgenic females and, in particular, males, with no changes in CB₁ receptors (Figure 1). In addition, the anandamide‐synthesizing enzyme NAPE‐PLD increased in SOD1^G93A transgenic males, although not in females (Figure 2), whereas DAGL, the 2‐arachidonoylglycerol‐synthesizing enzyme, was not altered in SOD1^G93A transgenic animals, although the probability levels were close to reach statistical significance in males (P = 0.078; Figure 2). Endocannabinoid degrading enzymes, FAAH and MAGL, were not significantly affected between SOD1^G93A transgenic mice compared to their littermate nontransgenic siblings (Figure 3). It is important to remark that SOD1^G93A transgenic mice showed, in some cases and for some parameters, a high variability, presumably related to differences in the degree of neurological deterioration (see below). This may explain that some differences do not reach statistical significance and remain as mere trends toward a change.

Figure 1.

Gene expression for CB ₁ and CB ₂ receptors measured in the spinal cord of male and female SOD‐1 transgenic or wild‐type mice (at 120 days after birth). Details in the text. Values correspond to % of change over wild‐type animals and are expressed as means ± SEM for 7–8 animals per group. Data were analyzed using unpaired Student's t‐test.

Figure 2.

Gene expression for NAPE‐PLD and DAGL enzymes measured in the spinal cord of male and female SOD‐1 transgenic or wild‐type mice (at 120 days after birth). Details in the text. Values correspond to % of change over wild‐type animals and are expressed as means ± SEM for 6–8 animals per group. Data were analyzed using unpaired Student's t‐test.

Figure 3.

Gene expression for FAAH and MAGL enzymes measured in the spinal cord of male and female SOD‐1 transgenic or wild‐type mice (at 120 days after birth). Details in the text. Values correspond to % of change over wild‐type animals and are expressed as means ± SEM for 7–8 animals per group. Data were analyzed using unpaired Student's t‐test.

Experiment II: Investigation of Sativex^® as a Disease‐Modifying Therapy in SOD1^G93A Mutant Mice

In the second objective of this study, we examined potential neuroprotective effects of the Sativex^®‐like combination of phytocannabinoids in SOD1^G93A transgenic mice. To this end, we treated males and females when first motor symptoms have appeared (9 weeks in the case of males and 10 weeks in females; see Figure 4) up to the end stage of the disease (around 130 days). We observed that the treatment with the Sativex^®‐like combination of phytocannabinoids slightly delayed the progression of neurological deficits in the early stages of the disease, in particular in females (Figure 4). It also tended to increase animal survival, an effect only observed in females (Figure 4), as well as it produced a partial recovery in the weight loss typical of transgenic animals, although this effect was seen only in males, not in females (data not shown). Nontransgenic littermate siblings treated with vehicle or Sativex^® did not present any differences in relation to the neurological score (constantly maintained at a value of 5), animal survival data, and weight recording, so their data were omitted from the figures to improve clarity.

Discussion

The first objective of the present study was to identify possible differences in the expression of endocannabinoid receptors and enzymes in the spinal cord between SOD1^G93A mutant mice and their nontransgenic littermate siblings. These differences may be of interest for a better design of future cannabinoid treatments targeting the altered endocannabinoid elements. To this end, we used SOD1^G93A transgenic animals at an end stage of the disease in which the neurological deterioration is evident and highly disabling. According to previous literature 21, 24 and also to our own data, the disease initiates in transgenic mice around 9–11 weeks after birth with some subtle differences between genders (males being affected earlier than females; see Figure 4). There is also certain degree of individual variability, with some animals being affected earlier than others, which is intrinsic to the hybrid B6SJL background of animals used here. The disease progresses up to moribundity that occurs at 19–20 weeks after birth 21, 24. For this objective, we used male and female transgenic mice at 17 weeks after birth, which presented a neurological score of 1.83 ± 0.33 (n = 12) in males and of 1.06 ± 0.31 (n = 11) in females, compared to their corresponding wild‐type animals of similar age and gender whose clinical score was always 5. These mice showed a marked up‐regulation of CB₂ receptors, presumably in glial elements, as has been found in experimental models of other chronic progressive disorders (reviewed in refs. 19 and 20). In fact, this up‐regulation was also found in ALS patients in a previous study conducted in postmortem spinal cord samples 18, as well as in experimental models 17. This type of response supports the activity of those cannabinoids targeting the CB₂ receptor as neuroprotective and antiinflammatory agents, a fact successfully demonstrated in the SOD1^G93A transgenic mouse model of ALS 16, 17. The novel aspect of our observation is that the up‐regulation of CB₂ receptors occurred in both genders, although it was more pronounced in the case of SOD1^G93A mutant males. These animals also exhibited other notable changes that were not found in females. For example, certain trends in MAGL (increase) and DAGL (decrease) enzymes, which would be compatible with the reduction in 2‐arachidonoyl‐glycerol levels seen by Witting et al. 21. In addition, the anandamide‐synthesizing enzyme NAPE‐PLD was significantly increased in SOD1^G93A transgenic males with no changes in the FAAH enzyme that degrades this endocannabinoid, which correlates with the increased levels of anandamide detected in the spinal cords of SOD1^G93A transgenic mice in the same study 21.

In addition to these biochemical data, the present study also provides additional evidence in support of cannabinoids as a possible neuroprotective therapy in ALS, as was indicated by some previous studies (reviewed in ref. 12). Given that most of these previous pharmacological studies used individual cannabinoid compounds although identified different potential targets (see ref. 12), we wanted to investigate a phytocannabinoid combination, the cannabis‐based medicine Sativex^®, with a broad spectrum of pharmacological actions. Sativex^® has been recently approved for the treatment of other neurological disorders 22, which may facilitate the clinical projection of the potential effects that may be found in this study. As mentioned above, Sativex^® may cover all pharmacological targets that have been found of interest in this experimental ALS model: (1) it contains Δ⁹‐THC, which was beneficial in previous studies 13 and is active at the CB₂ receptor, whose activation with a selective synthetic cannabinoid was also beneficial 16, 17; and (2) it also contains CBD, which had not been previously investigated in ALS, but it provides important antioxidant properties and also the possibility to inhibit FAAH enzyme (reviewed in ref. 25), which has been found to serve as a potential target in studies using genetic ablation or pharmacological inhibition 15. Our study demonstrated that Sativex^®‐like combination of phytocannabinoids was effective to delay disease progression in the initial stages of the disease, in particular in females, although the effects were lost during progression of the disease. We also quantified the animal survival using Kaplan–Meier curves, and although the results did not reach statistical significance, we could appreciate a trend toward an increase in the survival of females after the treatment with a Sativex^®‐like combination of phytocannabinoids. The fact that females were apparently more responsive to Sativex^® is intriguing and may be related to the differences in the hormonal status between both genders. In this sense, a recent study 26 has demonstrated: (1) that the treatment with Δ⁹‐THC increases pregnenolone synthesis in the brain and, to a lesser extent, in the spinal cord, through stimulation of CB₁ receptors; and (2) that this neurosteroid, which plays a critical role as precursor in the synthesis of different steroid compounds, serves as a negative allosteric modulator of CB₁ receptor signaling 26. It is well known that males and females present some differences in their requirements for neurosteroid biosynthesis, which may be in part responsible for the different levels of pregnenolone found in male and female brain areas, including the spinal cord 27. Therefore, it is possible that gender‐dependent differences in pregnenolone availability to inhibit CB₁ receptor signaling may explain that transgenic females are more responsive to Sativex^® than transgenic males, but this would require additional investigation. However, preliminary data obtained in our laboratory using analysis by Nissl staining of the spinal cord of SOD1^G93A transgenic mice euthanized at a late stage of the disease revealed that large motor neurons could be relatively preserved in those animals treated with this phytocannabinoid combination. However, they may not be functional given that this protection does not necessarily translate at the neurological level, as found in the present study, and these effects were similar in both genders. Follow‐up studies will have to confirm these preliminary data, but, if confirmed, this will mean that the protective effect of phytocannabinoids could affect primarily the survival of the motor neuron, whose cell body is stained with cresyl‐violet, but this level of protection would not be sufficient with the neuron‐muscle synapse, which appears to be significantly hampered in view of the weak clinical recovery. Anyway, our data support a relative efficacy of this treatment, although they also suggest the need to optimize it in follow‐up studies, for instance: (1) by using increasing doses of Sativex^®‐like combination of phytocannabinoids (despite that, dose used here is already high and, in the case of Δ⁹‐THC, has been found to be effective in previous studies 13); (2) by using a different combination of phytocannabinoids (e.g., a mixture with high Δ⁹‐THC and low CBD given that CBD may act as an antagonist for certain Δ⁹‐THC effects; see ref. 28); and (3) by using Sativex^®‐like combination of phytocannabinoids as an adjunctive therapy with other therapies used or investigated in ALS (e.g., riluzole).

In conclusion, our results demonstrated different changes in endocannabinoid signaling, in particular a marked up‐regulation of CB₂ receptors, in SOD1^G93A transgenic mice, and provide support that Sativex^® (or alternative Sativex^®‐like combinations of phytocannabinoids) may serve as a novel disease‐modifying therapy in ALS, a disorder with a poor therapeutic outcome at present with only one medicine already approved, Rilutek^®, but with a modest efficacy on disease progression. Anyway, more preclinical studies in additional models of ALS, i.e., TDP‐43 transgenic mice, will be necessary before testing the clinical efficacy of Sativex^® in ALS patients.

Conflict of Interest

The authors have formal links with GW Pharmaceuticals that funds some of their research.