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* Sobell Department of Motor Neuroscience and Movement Disorders and
Department of Neuroinflammation, Institute of Neurology, University College London, Queen Square, London, United Kingdom; and
Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples, Italy
1Correspondence: Sobell Department of Motor Neuroscience and Movement Disorders, University College London, Queen Square, London, WC1N 3BG, UK. E-mail: l.greensmith{at}ion.ucl.ac.uk
SPECIFIC AIMS
WE INVESTIGATED THE NEUROPROTECTIVE potential of cannabinoids in the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS) by using two different strategies: i) postsymptomatic treatment with an exogenous synthetic cannabinoid and ii) genetic augmentation of endocannabinoids. To elucidate the mechanism of action of cannabinoids, we genetically ablated the CB1 receptor in SOD1G93A mice. The effects of manipulating the cannabinoid system on disease progression and life span of SOD1G93A mice were then assessed.
PRINCIPAL FINDINGS
1. Endocannabinoid levels are up-regulated with disease progression in SOD1G93A mice
Levels of the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) were measured in spinal cords of wild-type (WT) and SOD1G93A mice at 40 (presymptomatic), 90 (symptomatic), and 120 d of age (late-stage). Endocannabinoid levels are up-regulated in the spinal cords of SOD1G93A mice but only after symptom onset. This up-regulation in endocannabinoids might be a protective response to counteract disease progression. To examine this possibility and to enhance this endogenous defense response, we manipulated the cannabinoid system in SOD1G93A mice by pharmacological and genetic means.
2. Postsymptomatic treatment with a synthetic cannabinoid, WIN55,212–2, delays disease progression in SOD1G93A mice
Transgenic SOD1G93A mice were treated with WIN55,212–2 (5 mg/kg, i.p.), a synthetic cannabinoid receptor agonist, from 90 d of age, after symptom onset. The effects on disease progression were assessed in vivo at 120 d, a late symptomatic stage of disease.
i) Muscle force
In untreated SOD1G93A mice, the TA and EDL hind-limb muscles are significantly weaker than in WT littermates (P<0.005). However, in SOD1G93A mice treated with WIN55,212–2, these muscles are significantly stronger than in untreated SOD1G93A littermates (P<0.05).
ii) Motor unit survival
The number of functional motor units in each EDL muscle was established in vivo by stimulating the sciatic nerve with stimuli of increasing intensity, which resulted in stepwise increments in twitch tension due to successive recruitment of motor axons. In WT mice at 120 d of age, EDL muscles have 29 (±0.9) motor units compared with only 12 (±0.9; P<0.001) in untreated SOD1G93A mice. Treatment with WIN55,212–2 significantly improves motor unit survival in SOD1G93A mice and 19 (±0.9; P<0.001) motor units survive at 120 d of age.
iii) Muscle fatigue characteristics
EDL is normally a fast muscle that fatigues rapidly when repeatedly stimulated. These characteristics change dramatically as disease progresses in SOD1G93A mice; in the late stages of disease, EDL becomes fatigue-resistant. However, treatment with WIN55,212–2 prevents this change, and EDL muscles retain their normal fatigue characteristics.
iv) Muscle histochemistry
The changes in EDL fatigue characteristics in SOD1G93A mice are reflected in the histochemical properties of the muscle fibers. In WT EDL muscles, most muscle fibers stain lightly for SDH, a marker of oxidative capacity. In contrast, in 120 d SOD1G93A mice, greater proportions of fibers stain darkly for SDH, indicating an increased oxidative capacity. Treatment with WIN55,212–2, however, prevents this change in muscle fiber phenotype.
v) Motoneuron survival
At 120 d, significant motoneuron degeneration has occurred in untreated SOD1G93A mice, and only 140 (±6.7) motoneurons survive compared with 369 (±12.9; P<0.001) in WT littermates. However, treatment with WIN55,212–2 rescues a significant proportion of motoneurons so that 42% more motoneurons survive at 120 d compared with untreated SOD1G93A littermates.
vi) Life span
Surprisingly, WIN55,212–2 has no effect on the life span of SOD1G93A mice, which live on average for 134 d (±2.3). This finding is not significantly different from untreated SOD1G93A littermates (P=0.96).
3. Genetic augmentation of endocannabinoid levels ameliorates disease in 90-day-old SOD1G93A mice
In view of the delay in disease progression in WIN55,212–2-treated SOD1G93A mice, the effect of elevating endogenous cannabinoids was assessed by genetically ablating the Faah enzyme in SOD1G93A mice (SOD1.Faah –/–). Furthermore, to elucidate the mechanism of action of cannabinoids, the CB1 receptor was ablated in SOD1G93A mice (SOD1.Cnr1 –/–). The effects of these genetic manipulations were assessed in vivo at 90 d of age.
i) Muscle force
In 90 d SOD1G93A mice, TA and EDL muscles are already substantially weaker than in WT littermates. However, in SOD1.Faah –/– mice, we found no reduction in muscle force and TA and EDL are as strong as in WT mice. In contrast, ablation of the CB1 receptor does not maintain muscle force in SOD1G93A mice (P>0.1).
ii) Motor unit survival
In SOD1G93A mice, a significant number of motor units have died by 90 d and only 19 (±1.4) motor units survive compared with 30 (±1.2; P<0.001) motor units in WT littermates. However, no motor unit loss occurs in SOD1.Faah –/– mice at this age and 27 (±1.5; P<0.001) motor units survive. In contrast, in SOD1.Cnr1 –/– mice, motor units are not rescued and, similar to SOD1G93A mice, only 19 (±1.4; P>0.7) motor units survive.
iii) Motoneuron survival
Motoneuron survival was assessed in spinal cords of WT, SOD1G93A, SOD1.Faah –/– and SOD1.Cnr1 –/– mice. Significant motoneuron death occurs by 90 d in SOD1G93A mice, and only 237 (±19.5) motoneurons survive compared with 358 (±13.8; P<0.001) in WT littermates. In SOD1G93A mice in which the Faah enzyme was ablated, little motoneuron death occurs by 90 d, and 305 (±18.1) motoneurons survive in SOD1.Faah –/– mice (P=0.035). However, in SOD1.Cnr1 –/– mice the absence of the CB1 receptor does not affect motoneuron survival and only 217 (±12.8; P=0.575) motoneurons survive at 90 d.
4. Ablation of the CB1 receptor extends life span in SOD1G93A mice
Surprisingly, despite the dramatic improvement in disease signs observed at 90 d, ablation of the Faah enzyme has no significant effect on life span (P=0.399). Conversely, although no effect on disease progression was found at 90 d in SOD1.Cnr1 –/– mice, these mice showed a significant increase in life span (P=0.03). Thus, SOD1.Cnr1 –/– mice live on average for 133 d compared with 118 d in SOD1G93A mice, an increase of 13%.
CONCLUSIONS AND SIGNIFICANCE
Increasing evidence suggests that cannabinoids might have therapeutic potential in neurodegenerative conditions. In a variety of in vivo and in vitro models, cannabinoids exert neuroprotective effects under excitotoxic, ischemic, and inflammatory conditions. This combination of neuroprotective actions might be particularly relevant to ALS and suggests that cannabinoids might have a greater impact on disease progression than the established therapy that targets excitotoxicity alone.
As a consequence of disease progression in SOD1G93A mice, levels of endocannabinoids are up-regulated. This up-regulation might represent a neuroprotective response. However, such an endogenous response is clearly not robust enough to counteract disease progression. Therefore, in this study we aimed to enhance this endogenous protective mechanism in SOD1G93A mice using two methods: i) treatment with a synthetic exogenous cannabinoid and ii) genetic augmentation of endocannabinoid levels. Treatment with the synthetic cannabinoid, WIN55,212–2, significantly delays disease progression in SOD1G93A mice. Furthermore, genetic ablation of the Faah enzyme and the consequent elevation in AEA levels exert a much stronger neuroprotective action and, at 90 d of age, essentially ameliorated signs of disease in SOD1G93A mice. However, these improvements in SOD1G93A mice at 90 d were not reflected in an increased life span. These findings suggest that, although endocannabinoids may be of significant therapeutic benefit, their neuroprotective actions are confined mainly to the early stages of disease. This study, therefore, identifies the Faah enzyme as a novel therapeutic target in ALS and suggests that Faah inactivation might be particularly effective if used in combination with agents that act on pathological mechanisms active during later stages of the disease.
Cannabinoids might exert their neuroprotective actions via activation of CB1 or CB2 receptors (Fig. 1
), although CB-receptor independent effects might also contribute. In this study, however, we have shown that ablation of the CB1 receptor significantly extended the life span of SOD1G93A mice, despite having no effect on disease progression at 90 d of age. This finding suggests that CB1 receptor activation might not play a significant role in the cellular defense mechanism of motoneurons in SOD1G93A mice. Furthermore, the neuroprotective effects observed following pharmacological and genetic augmentation of cannabinoid levels are not necessarily mediated by the CB1 receptor, and indeed inhibition of the CB1 receptor might actually be neuroprotective. Therefore, in contrast to previous studies that have suggested that cannabinoids exert neuroprotection via the CB1 receptor, the present results suggest that activation of CB2 receptors might underlie the beneficial effects of cannabinoids at least in SOD1G93A mice (Fig. 1)
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In conclusion, our results show that increased endocannabinoid tone, obtained either following treatment with synthetic cannabinoids or by ablation of the Faah enzyme, ameliorates disease, at least in the short term. These neuroprotective effects appear to be mediated by a non-CB1 receptor-dependent mechanism. Therefore, further studies are needed to increase our understanding of the therapeutic potential of the Faah enzyme and the CB2 receptor in ALS.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4743fje
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