Alzheimer’s and Cannabis; How Marijuana Affects AD
Alzheimer’s disease (AD) is the most common form of dementia among older people. Dementia is a brain disorder that seriously affects a person’s ability to carry out daily activities. AD begins slowly. It first involves the parts of the brain that control thought, memory and language.
Recent years have brought a wealth of new scientific understanding regarding how medical marijuana cannabis can be beneficial for treating Alzheimers.
Alzheimer’s disease (AD) is a neurological disorder of unknown origin that is characterized by a progressive loss of memory and learned behavior. Patients with Alzheimer’s are also likely to experience depression, agitation, and appetite loss, among other symptoms. Over 4.5 million Americans are estimated to be afflicted with the disease. No approved treatments or medications are available to modulate the progression of AD, and few pharmaceuticals effectively treat symptoms of the disease.
Preclinical data shows the potential of cannabinoids to moderate the progression of AD while clinical data demonstrates that these compounds can provide symptom relief.
Writing in the Journal of Neuroscience, investigators at Madrid’s Complutense University and the Cajal Institute in Spain reported that the intracerebroventricular administration of the synthetic cannabinoid WIN 55,212-2 prevented cognitive impairment and decreased neurotoxicity in rats injected with amyloid-beta peptide (a protein believed to induce Alzheimer’s). Additional synthetic cannabinoids were also found to reduce the inflammation associated with Alzheimer’s disease in human brain tissue in culture. “Our results indicate that … cannabinoids succeed in preventing the neurodegenerative process occurring in the disease,” investigators concluded. Follow up studies by investigators demonstrated that the administration of the nonpsychotropic plant cannabinoid cannabidiol also mitigated memory loss in a mouse model of the disease.
Investigators at The Scripps Research Institute in California have reported that THC administration inhibits the enzyme responsible for the aggregation of amyloid plaque — the primary marker for Alzheimer’s disease — in a manner “considerably superior” to approved AD drugs such as donepezil and tacrine. “Our results provide a mechanism whereby the THC molecule can directly impact Alzheimer’s disease pathology,” researchers concluded. “THC and its analogues may provide an improved therapeutic [option] for Alzheimer’s disease [by]… simultaneously treating both the symptoms and the progression of [the] disease.” Investigators at the Salk Institute in 2016 reported similar findings in a series of exploratory studies.
The administration of both THC and synthetic cannabinoid agonists have been shown to influence memory loss in animal models. For example, investigators at Ohio State University, Department of Psychology and Neuroscience, reported that older rats administered daily doses of WIN 55,212-2 for a period of three weeks performed significantly better than non-treated controls on a water-maze memory test. Writing in the journal Neuroscience, they reported that rats treated with the compound experienced a 50 percent improvement in memory and a 40 to 50 percent reduction in inflammation compared to controls. Israeli researchers in 2017 reported that THC administration can reverse age-related memory impairment in rats, and may offer a potential treatment option in patients with dementia and other neurodegenerative illnesses.
Previous preclinical studies have demonstrated that cannabinoids can prevent neuronal cell death. Some experts believe that these neuroprotective properties could play a role in moderating AD. Writing in the British Journal of Pharmacology, investigators at Ireland’s Trinity College Institute of Neuroscience concluded, “[C]annabinoids offer a multi-faceted approach for the treatment of Alzheimer’s disease by providing neuroprotection and reducing neuroinflammation, whilst simultaneously supporting the brain’s intrinsic repair mechanisms by augmenting neurotrophin expression and enhancing neurogenesis. … Manipulation of the cannabinoid pathway offers a pharmacological approach for the treatment of AD that may be efficacious than current treatment regimens.”
Clinical trials demonstrate that cannabinoid therapy can mitigate certain AD symptoms. For instance, investigators at Berlin Germany’s Charite Universitatmedizin, Department of Psychiatry and Psychotherapy, reported that the daily administration of 2.5 mg of synthetic THC over a two-week period reduced nocturnal motor activity and agitation in AD patients in an open-label pilot study.
Clinical data presented at the 2003 annual meeting of the International Psychogeriatric Association reported that the oral administration of up to 10 mg of synthetic THC reduced agitation and stimulated weight gain in late-stage Alzheimer’s patients in an open-label clinical trial. Improved weight gain and a decrease in negative feelings among AD patients administered cannabinoids were previously reported by investigators in the International Journal of Geriatric Psychiatry in 1997.
Most recently, Israeli researchers assessed the safety and efficacy of THC-infused oil in Alzheimer’s patients in a four-week trial. Participants experienced decreased incidences of delusions, agitation, irritability, and apathy following treatment. Their quality of sleep also improved. “Adding medical cannabis oil to AD patients’ pharmacotherapy is safe and a promising treatment option,” investigators concluded.
 Ramirez et al. 2005. Prevention of Alzheimer’s disease pathology by cannabinoids. The Journal of Neuroscience 25: 1904-1913.
 Israel National News. December 16, 2010. “Israeli research shows cannabidiol may slow Alzheimer’s disease.”
 Eubanks et al. 2006. A molecular link between the active component of marijuana and Alzheimer’s disease pathology. Molecular Pharmaceutics 3: 773-777.
 Salk News. June 27, 2016. “Cannabinoids remove plaque-forming Alzheimer’s proteins from brain cells“
 Marchalant et al. 2007. Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic brain inflammation. Neuroscience 144: 1516-1522.
 Science Daily. May 8, 2017. “Cannabis reverses aging process in the brain, study suggests.”
 Hampson et al. 1998. Cannabidiol and delta-9-tetrahydrocannabinol are neuroprotective antioxidants. Proceedings of the National Academy of Sciences 95: 8268-8273.
 Campbell and Gowran. 2007. Alzheimer’s disease; taking the edge off with cannabinoids? British Journal of Pharmacology 152: 655-662.
 Walther et al. 2006. Delta-9-tetrahydrocannabinol for nighttime agitation in severe dementia. Physcopharmacology 185: 524-528.
 Volicer et al. 1997. Effects of dronabinol on anorexia and disturbed behavior in patients with Alzheimer’s disease. International Journal of Geriatric Psychiatry 12: 913-919.
 Shelef et al. 2016. Safety and efficacy of medical cannabis oil for behavioral and psychological symptom of dementia: An open label, add-on, pilot study. Journal of Alzheimer‘s Disease 51: 15-19.
Neuroprotective effect of cannabidiol, a non-psychoactive component from Cannabis sativa, on beta-amyloid-induced toxicity in PC12 cells.
- Department of Experimental Pharmacology, University of Naples Federico II, Naples, Italy.
Alzheimer’s disease is widely held to be associated with oxidative stress due, in part, to the membrane action of beta-amyloid peptide aggregates. Here, we studied the effect of cannabidiol, a major non-psychoactive component of the marijuana plant (Cannabis sativa) on beta-amyloid peptide-induced toxicity in cultured rat pheocromocytoma PC12 cells. Following exposure of cells to beta-amyloid peptide (1 micro g/mL), a marked reduction in cell survival was observed. This effect was associated with increased reactive oxygen species (ROS) production and lipid peroxidation, as well as caspase 3 (a key enzyme in the apoptosis cell-signalling cascade) appearance, DNA fragmentation and increased intracellular calcium. Treatment of the cells with cannabidiol (10(-7)-10(-4)m) prior to beta-amyloid peptide exposure significantly elevated cell survival while it decreased ROS production, lipid peroxidation, caspase 3 levels, DNA fragmentation and intracellular calcium. Our results indicate that cannabidiol exerts a combination of neuroprotective, anti-oxidative and anti-apoptotic effects against beta-amyloid peptide toxicity, and that inhibition of caspase 3 appearance from its inactive precursor, pro-caspase 3, by cannabidiol is involved in the signalling pathway for this neuroprotection.
In vivo Evidence for Therapeutic Properties of Cannabidiol (CBD) for Alzheimer’s Disease
Alzheimer’s disease (AD) is a debilitating neurodegenerative disease that is affecting an increasing number of people. It is characterized by the accumulation of amyloid-β and tau hyperphosphorylation as well as neuroinflammation and oxidative stress. Current AD treatments do not stop or reverse the disease progression, highlighting the need for new, more effective therapeutics. Cannabidiol (CBD) is a non-psychoactive phytocannabinoid that has demonstrated neuroprotective, anti-inflammatory and antioxidant properties in vitro. Thus, it is investigated as a potential multifunctional treatment option for AD. Here, we summarize the current status quo of in vivo effects of CBD in established pharmacological and transgenic animal models for AD. The studies demonstrate the ability of CBD to reduce reactive gliosis and the neuroinflammatory response as well as to promote neurogenesis. Importantly, CBD also reverses and prevents the development of cognitive deficits in AD rodent models. Interestingly, combination therapies of CBD and Δ9-tetrahydrocannabinol (THC), the main active ingredient of cannabis sativa, show that CBD can antagonize the psychoactive effects associated with THC and possibly mediate greater therapeutic benefits than either phytocannabinoid alone. The studies provide “proof of principle” that CBD and possibly CBD-THC combinations are valid candidates for novel AD therapies. Further investigations should address the long-term potential of CBD and evaluate mechanisms involved in the therapeutic effects described.
Alzheimer’s Disease (AD) is a debilitating neurodegenerative disease that is characterized by cognitive decline. It is the most common form of dementia, accounting for over 60% of cases and affecting over 33 million people worldwide (Wisniewski and Goni, 2014; Alzheimer’s Association, 2015). Unfortunately, as a result of the aging population, this number is expected to reach 115 million by the year 2050 (Wisniewski and Goni, 2014). AD typically begins with mild deficits in short-term memory, learning, communication and spatial orientation. In the moderate stage of the disease, the deficits begin to affect everyday life including eating, dressing and emotional control (Alzheimer’s Association, 2015). In the late stages of the disease there is global disruption of cognitive ability, with severe impairments in speech and facial recognition, all of which renders the patients in need of 24-h care. As the disease progresses, patients become increasingly susceptible to other illnesses as well (Alzheimer’s Association, 2015).
AD is classified into two types, late-onset sporadic AD (>95% of cases) or early-onset familial AD (<5% cases) (Gotz and Ittner, 2008). Although, sporadic AD is the most common form, it is much less understood than familial AD. Familial AD is also known as the genetic form, as it results from autosomal dominant mutations in the amyloid precursor protein (APP) gene or in the presenilin 1 and 2 (PS1 and PS2) genes (Gotz and Ittner, 2008; Bettens et al., 2013). APP is the precursor molecule, which is cleaved into amyloid-β (Aβ) peptides, while PS1 and PS2 encode the γ-secretase and β-secretase complexes that mediate APP splicing (Bettens et al., 2013; Gotz and Ittner, 2008). After APP splicing Aβ can exist in two forms, Aβ40 and Aβ42. Aβ42 is thought to be the more toxic form of the protein as it aggregates more readily than Aβ40 (Chapman et al., 2001). The cause of sporadic AD is less clear and yet to be defined, however, recent research indicates that it may result from a complex interaction between several environmental factors and various susceptible genes. Numerous genes have been reported as susceptible genes for sporadic AD with the best-documented one being APOE (Kamboh, 2004).
Although familial and sporadic AD differ in their cause, the progression of the disease from this point onwards appears to be the same. Both forms of AD exhibit a neurodegenerative cascade that appears to be instigated by the accumulation of Aβ (forming senile plaques) and hyperphosphorylated tau [forming neurofibrillary tangles (NFTs)] (Chapman et al., 2001). The cascade induces neuroinflammation and oxidative stress, which creates a neurotoxic environment that potentiates neurodegeneration and eventually leads to cognitive decline (Hardy and Selkoe, 2002; Ahmed et al., 2015). Also, Aβ-induced neurodegeneration elevates glutamate levels in the cerebral spinal fluid of AD patients (Pomara et al., 1992) and cholinergic neurons are lost in brain areas relevant for memory processing (and accompanied by a decrease in acetylcholine) (Schliebs and Arendt, 2011).
Despite the increase in our understanding of disease mechanism, the current approved AD treatments only provide limited therapeutic benefits. There are four approved drugs available, three are acetylcholinesterase inhibitors (rivistagmine, donepezil and galantamine) and one is a N-methyl-D-aspartate (NMDA) receptor antagonist (memantine) (Mangialasche et al., 2010). Unfortunately, all of them have been associated with adverse effects. Acetylcholinesterase inhibitors may cause nausea, vomiting, diarrhea and weight loss (Kaduszkiewicz et al., 2005), while memantine is known to cause hallucinations, dizziness and fatigue (Herrmann et al., 2011). Furthermore, none of these treatments prevent or reverse the progression of the disease but rather they treat the disease symptoms with limited efficacy (Salomone et al., 2012).
Current clinical trials to evaluate new AD treatments are targeting various aspects of AD pathology, with a strong focus on Aβ. Clinical trials have investigated both β- and γ-secretase inhibitors, which play a crucial role in the formation of pathological Aβ. Unfortunately, β-secretases are difficult to target and γ-secretases have a wide range of functions resulting in adverse side effects (e.g., impaired cognition and functionality, gastrointestinal toxicity and increased incidence of skin cancer) (Imbimbo and Giardina, 2011; Schenk et al., 2012). Active and passive immunotherapies to target senile plaques and NFTs have also been investigated. Aβ immunotherapies in mouse models demonstrated potential as they increased microglial phagocytosis of Aβ and reduced cognitive decline. However, in phase II and III clinical trials those therapies have demonstrated limited efficacy or resulted in severe adverse effects (e.g., meningoenchephalitis) (Mullane and Williams, 2013). A recent study investigating an antibody based immunotherapy for Aβ found promising results in phase I and phase II trials but this therapy is yet to undergo phase III clinical trials (Sevigny et al., 2016). Tau immunotherapies were effective in AD mouse models but have provided limited success in clinical trials (McGeer et al., 2006; Schenk et al., 2012; Mullane and Williams, 2013).
Epidemiological data have shown that non-steroidal anti-inflammatory drugs (NSAIDs) are associated with a reduced risk of AD (McGeer et al., 2006). Furthermore, animal studies indicated that NSAID treatment could attenuate AD pathogenesis, proposing that inhibiting neuroinflammation may slow the progression of AD (Maccioni et al., 2009). However, NSAIDs have also been associated with severe long-term adverse effects (e.g., gastrointestinal problems) and have only shown limited efficacy in reducing or preventing clinical symptoms (McGeer et al., 2006; Rojo et al., 2008).
It is unlikely that any drug acting on a single pathway or target will mitigate the complex pathoetiological cascade leading to AD. Therefore, a multifunctional drug approach targeting a number of AD pathologies simultaneously will provide better, wider-ranging benefits than current therapeutic approaches (Van der Schyf and Geldenhuys, 2011; Bedse et al., 2015). Importantly, the endocannabinoid system has recently gained attention in AD research as it is associated with regulating a variety of processes related to AD, including oxidative stress (Marsicano et al., 2002), glial cell activation (Germain et al., 2002) and clearance of macromolecules (Bilkei-Gorzo, 2012).
The phytocannabinoid cannabidiol (CBD) is a prime candidate for this new treatment strategy. CBD has been found in vitro to be neuroprotective (Esposito et al., 2006b), to prevent hippocampal and cortical neurodegeneration (Hamelink et al., 2005), to have anti-inflammatory and antioxidant properties (Mukhopadhyay et al., 2011), reduce tau hyperphosphorylation (Esposito et al., 2006a) and to regulate microglial cell migration (Walter et al., 2003; Martín-Moreno et al., 2011). Furthermore, CBD was shown to protect against Aβ mediated neurotoxicity and microglial-activated neurotoxicity (Janefjord et al., 2014), to reduce Aβ production by inducing APP ubiquination (Scuderi et al., 2014) and to improve cell viability (Harvey et al., 2012) (summarized in Table Table1).1). These properties suggest that CBD is perfectly placed to treat a number of pathologies typically found in AD. In the following, we will outline in brief the endocannabinoid system and the pharmacological profile of CBD before discussing recent advances in the evaluation of the therapeutic properties of CBD (and CBD-THC combinations) using in vivo AD rodent models.
The endocannabinoid system and CBD pharmacology
The endocanabinoid system (eCBS) consists of endocannabinoids [e.g., anandamide and 2-arachiodonoylglycerol (2-AG)], enzymes required for their synthesis and degradation [fatty acid amide hydrolase (FAAH), monoglyceride lipase (MAGL), and diacylglycerol lipase (DAGL)], and cannabinoid receptors [the best described being cannabinoid receptors 1 and 2 (CB1 and CB2)], (Di Marzo et al., 2015). Post mortem analyses have found that several of these components are altered in both composition and signaling in AD postmortem brain tissue (Aso and Ferrer, 2015).
CBD has a complex interaction with the eCBS. It has demonstrated low displacement at the CB1 and CB2 receptors compared to other cannabinoids such as Δ9-tetrahydrocannabinol (THC) (Thomas et al., 1998). CBD has also been shown to have low affinity for both cannabinoid receptors (Petitet et al., 1998) and has antagonistic properties against the synthetic cannabinoid, CP 55 940, which is a potent agonist at both CB1 and CB2 receptors. Interestingly, CBD antagonizes CP 55, 940 at a much lower concentration than it binds to the cannabinoid receptors, suggesting it may act at a prejunctional site which is not the cannabinoid receptors (Pertwee et al., 2002). CBD acts as an inverse agonist at the CB2 receptors, which may explain some of its anti-inflammatory properties as inverse agonists at CB2 receptors are able to inhibit the migration of immune cells (Lunn et al., 2006). CBD has also been found to act as an antagonist at the cannabinoid G-protein receptors (GPR) GPR55 and GPR18 (Ryberg et al., 2007; McHugh et al., 2010), as well as activate the putative abnormal CBD receptor (Pertwee, 2008) and the vanilloid receptor 1 (Bisogno et al., 2001). Finally, CBD interacts with various neurotransmitter systems including glutamate receptors [i.e., NMDA receptors, 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid (AMPA) receptors and kainite receptors] and the serotonergic receptor, 5-HT1A (Russo et al., 2005). The wide range of targets of CBD emphasizes its potential as a multimodal drug for AD treatment.
CBD effects in pharmacological rodent models of AD
The in vivo therapeutic potential of CBD in AD has not been widely documented, however, there are a number of studies that have reported the effect of CBD in pharmacological models of AD (e.g., inoculation with fibrillar Aβ). These studies have described anti-inflammatory and neuroprotective effects of CBD. The in vivo anti-inflammatory effects of CBD were confirmed in a mouse model of AD where the mice were intrahippocampally injected with human Aβ42 and then treated daily with intraperitoneal (i.p.) injections of CBD (2.5 or 10 mg/kg) for 7 days (Esposito et al., 2007). The results from this study demonstrated that CBD was able to dose-dependently inhibit glial fibrillary acidic protein (GFAP) mRNA and protein expression. GFAP is the best known marker of activated astrocytes and thought to be one of the main features of reactive gliosis (Esposito et al., 2007). Therefore, these results imply that CBD is able to reduce Aβ-induced reactive gliosis. In addition, CBD reduced both iNOS and interleukin-1β (IL-1β) protein expression and the related NO and IL-1β release (Esposito et al., 2007). NO and IL-1β are a few of the many active substances released by Aβ-stimulated microglia and therefore have been identified as potential modulators of neuronal damage. NO is a free radical and important in neuroinflammatory and neurodegenerative conditions, which include accelerating protein nitration and increasing tau hyperphosphorylation (Esposito et al., 2007). IL-1β is involved in the cytokine cycle responsible for neurodegeneration, the synthesis and processing of APP, the activation of astrocytes and the overexpression of iNOS and overproduction of NO (Esposito et al., 2007). Data from in vitro studies suggest that CBD may be able to reduce iNOS protein expression and NO release as a result of its ability to rescue the Wnt/β-catenin pathway, which plays a role in tau hyperphosphorylation (Esposito et al., 2006a). Finally, the ability of CBD to attenuate reactive gliosis may result from CBD’s ability to act as an inverse agonist at the cannabinoid receptor 2 (CB2), which is thought to be involved in reactive gliosis (Walter and Stella, 2004; Thomas et al., 2007).
The anti-inflammatory and neuroprotective effects of CBD were further investigated in a rat model of AD-related neuroinflammation. This study evaluated the involvement of the peroxisome proliferator activated receptor (PPAR) receptors in the therapeutic effects of CBD, as PPAR-γ receptors are increased in AD patients (Esposito et al., 2011). Adult, male rats were inoculated with human Aβ42 in the hippocampus and then treated with CBD (10 mg/kg) either in the presence or in the absence of a PPAR-γ or PPAR-α receptor antagonist for 15 days. CBD was able to dose-dependently decrease Aβ-induced expression of iNOS, GFAP, S100 calcium binding protein B (S100B) and p50 and p56 antibodies in rat astrocytes (Esposito et al., 2011). iNOS and GFAP, as mentioned previously, are key elements in reactive gliosis and therefore their reduction demonstrates CBD’s anti-inflammatory properties. CBD’s ability to reduce reactive gliosis is further emphasized by the inhibition of S100B. S100B is an astroglial-derived neurotrophin that plays a crucial role in the pro-inflammatory cytokine cycle and the promotion of APP to cleave Aβ42. It is also involved in the disruption of the Wnt/β-catenin pathway and therefore inhibits tau hyperphosphorylation (Esposito et al., 2011). Furthermore, the reduction of p50 and p56 expression indicates CBD’s ability to inhibit NF-κB and therefore emphasizes the responsibility of both PPAR-γ and NF-κB in CBD’s anti-inflammatory properties (Esposito et al., 2011). The therapeutic benefit of CBD was blocked when co-administered with the PPAR-γ antagonist (but not the PPAR-α antagonist) (Esposito et al., 2011), suggesting that CBD-induced anti-inflammatory properties are mediated (at least partially) through the PPAR-γ receptor (Esposito et al., 2011). Finally, the study found that CBD was able to restore CA1 pyramidal neurons to a similar integrity to that of the control rats. CBD also down-regulated gliosis and repaired neurogenesis in the dentate gyrus (Esposito et al., 2011).
One study to date has investigated the effects of CBD on cognition in a pharmacological model of AD. Three-month old mice were intraventricularly injected with 2.5 μg of fibrillar Aβ. They were then treated with 20 mg/kg CBD using daily i.p. injections for 1 week and then 3 times/week for the following 2 weeks. The spatial learning of the mice was then assessed in the Morris Water Maze (Martín-Moreno et al., 2011). CBD treatment was able to reverse the cognitive deficits of Aβ-treated mice. Interestingly, selective CB2 agonists did not prevent the cognitive deficit, indicating that CBD exerts this therapeutic effect via other mechanisms (Martín-Moreno et al., 2011). CBD treatment also prevented Aβ-induced IL-6 gene expression suggesting that the behavioral benefits documented may be mediated by glial activation modulation. However, CBD did not influence TNF-α gene expression. In vitro results from this study supported this finding as CBD treatment prevented the ATP-induced intracellular calcium increase and promoted microglial activation in cultured microglia (Martín-Moreno et al., 2011).
CBD effects in transgenic mouse models of AD
Although pharmacological models of AD are useful in producing AD-like symptoms, it is necessary to investigate the effects of CBD in transgenic mouse models as they result from gene mutations, which are seen in familial AD (e.g., APP, PS1, and PS2 gene mutations). Furthermore, based on the pharmacological protocols used, some effects of CBD could be related to a direct effect of the phytocannabinoid on exogenous Aβ administration rather than the long-term effects of the accumulated Aβ. Initially, two studies were conducted in our laboratories to elucidate the remedial and preventative potential of chronic CBD treatment in AD transgenic mice. To assess the remedial effects of CBD, adult male APPxPS1 mice were treated for 3 weeks with CBD (20 mg/kg CBD, daily i.p. injections) post onset of cognitive deficits and AD pathology (Cheng et al., 2014a). CBD treatment was able to reverse cognitive deficits in object recognition memory and social recognition memory without influencing anxiety parameters (Cheng et al., 2014a).
In the preventative treatment study, male APPxPS1 mice at the age of 2.5 months were treated for 8 months with either 20 mg/kg CBD or vehicle pellets using a daily voluntary oral administration protocol (Cheng et al., 2014b). This assessed the long-term effect of CBD prior to “AD onset.” Long-term CBD treatment was able to prevent the development of social recognition memory deficits without affecting anxiety domains in AD transgenic mice (Cheng et al., 2014b). These beneficial effects were not associated with a reduction in Aβ load or oxidative damage. There was also no difference in hippocampal or cortical soluble and insoluble levels of Aβ40 and Aβ42 in the AD transgenic mice regardless of treatment. Furthermore, cortical lipid oxidation levels were not altered by CBD treatment. However, the study did report a complex interaction between CBD treatment, AD genotype and cholesterol and phytosterol levels, suggesting they may be involved in the mechanisms behind the beneficial effects of CBD. There was also a subtle impact of CBD on inflammatory markers of the brain (Cheng et al., 2014b). Further research will be necessary to elucidate the potential mechanisms further, thereby also considering other treatment designs (i.e., different ages at treatment onset and CBD doses).
Recent research has indicated that a combination of CBD and Δ9-tetrahydrocannabinol (THC) can avoid the detrimental effects caused by THC-induced activation of the CB1 receptors (e.g., psychoactivity), and actually provide greater therapeutic benefits than either phytocannabinoid alone. Importantly, there is controversy about what the ratios of CBD:THC should be used in order to antagonize detrimental THC effects. It has been reported that a >10-fold higher dose of CBD was necessary to prevent the unwanted side effects of THC. Other research suggests that CBD may even modestly potentiate THC’s psychoactive effects (Fadda et al., 2004; Klein et al., 2011). Nevertheless, Sativex (GW pharmaceuticals, Salisbury, United Kingdom), a combination therapy using a 1:1 ratio of CBD and THC is approved as an anti-inflammatory drug treatment against spasms in multiple sclerosis and does not appear to be associated with any adverse THC effects, suggesting that CBD effectively blocks those at the ratio chosen (Collin et al., 2010; Novotna et al., 2011).
Three studies to date have evaluated the efficacy of a combination of CBD and THC on AD-related processes in vivo. The first study conducted by Casarejos et al. (2013) investigated the effects of Sativex in a mouse model of tauopathy. This mouse model was foremost a model of frontotemporal dementia, parkinsonism and lower motor neuron disease. The study found that Sativex decreased gliosis, increased the ratio of reduced/oxidized glutathione and reduced the levels of iNOS (Casarejos et al., 2013), thereby showing neuroprotective and anti-oxidant properties. Importantly, Sativex reduced Aβ and tau deposition in the hippocampus and cerebral cortex as well as increasing autophagy (Casarejos et al., 2013), thus implying, that although the mouse model is not directly related to AD, the therapeutic benefits are.
The second study conducted by Aso et al. (2015) compared the effect of CBD, THC and a CBD-THC combination in the APPxPS1 mouse model, in the early symptomatic phase (~6 months). This study found that all treatments improved memory deficits in the two-object recognition task but only the CBD-THC combination prevented the learning deficit seen in the active avoidance task. CBD-THC combination also decreased soluble Aβ42 levels and changed plaque composition while CBD and THC individually did not (Aso et al., 2015). Finally, reduced astrogliosis, microgliosis and inflammatory related molecules were more pronounced after treatment with the CBD-THC combination than either phytocannabinoid individually (Aso et al., 2015). This suggests that when CBD and THC are combined there may be either a summative effect or an interaction effect between the compounds, which potentiates their therapeutic-like effects (Aso et al., 2015). In this context, it should be mentioned, that although all treatments had cognition-improving characteristics in the object recognition task, THC alone had a detrimental effect on cognition in control mice, highlighting the need to be cautious when considering THC as a therapeutic. However, control mice treated with CBD-THC combination did not show any cognitive deficits suggesting that CBD may be able to antagonize the detrimental effects of THC (Aso et al., 2015).
In a very recent follow-up study, Aso et al. also investigated the effect of CBD-THC combination treatment on memory and brain pathology in aged male APPxPS1 mice and littermate controls (12 months) as well as non-aged controls, 3 months old control mice (Aso et al., 2016). Compared to the non-aged controls, vehicle-treated aged mice demonstrated impaired cognition in the two-object recognition task. Interestingly, CBD-THC combination restored the memory deficit of APPxPS1 but not WT control mice (Aso et al., 2016). In comparison to their previous study testing younger APPxPS1 mice (Aso et al., 2015), CBD-THC combination did not influence the Aβ load or the related glial reactivity in aged AD transgenic mice (Aso et al., 2016)., However, the combination treatment normalized synaptosome associated protein 25, glutamate receptors 2 and 3 and γ-aminobutyric acid receptor A subunit α1 expression, implying that CBD-THC may exert its beneficial effects on cognition via these mechanisms.
AD is a debilitating neurodegenerative disease that is becoming increasingly common in today’s society. Unfortunately, there is still no effective treatment that stops or reverses the disease progression. The studies reviewed in this mini review provide “proof of principle” for the therapeutic benefits CBD and possibly CBD-THC combinations pose for AD therapy (summarized in Table Table1).1). However, further dose-dependent investigations into transgenic mouse models of AD are necessary to understand the full potential and the long-term effects of CBD. Importantly, many of the discussed studies were conducted in mice aged between 3 and 6 months, which is quite young considering AD diagnosis is usually relatively late in the disease progression. Furthermore, it is necessary to investigate the effects of CBD in tauopathy mouse models specific to AD and in female mouse models as all studies reviewed were conducted in male mice only. Nevertheless, the studies discussed here provide promising preliminary data and the translation of this preclinical work into the clinical setting could be realized relatively quickly: CBD is readily available, appears to only have limited side effects (Bergamaschi et al., 2011) and is safe for human use (Leweke et al., 2012).
A Molecular Link Between the Active Component of Marijuana and Alzheimer’s Disease Pathology
Lisa M. Eubanks†, Claude J. Rogers†, Albert E. Beuscher IV‡, George F. Koob§, Arthur J. Olson‡, Tobin J. Dickerson†, and Kim D. Janda† Departments of Chemistry, Immunology, Molecular Biology, Molecular and Integrated Neurosciences Department (MIND), The Skaggs Institute for Chemical Biology, and Worm Institute for Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. email@example.com
Abstract Alzheimer’s disease is the leading cause of dementia among the elderly, and with the ever-increasing size of this population, cases of Alzheimer’s disease are expected to triple over the next 50 years. Consequently, the development of treatments that slow or halt the disease progression have become imperative to both improve the quality of life for patients as well as reduce the health care costs attributable to Alzheimer’s disease. Here, we demonstrate that the active component of marijuana, Δ9-tetrahydrocannabinol (THC), competitively inhibits the enzyme acetylcholinesterase (AChE) as well as prevents AChE-induced amyloid β-peptide (Aβ) aggregation, the key pathological marker of Alzheimer’s disease. Computational modeling of the THC-AChE interaction revealed that THC binds in the peripheral anionic site of AChE, the critical region involved in amyloidgenesis. Compared to currently approved drugs prescribed for the treatment of Alzheimer’s disease, THC is a considerably superior inhibitor of Aβ aggregation, and this study provides a previously unrecognized molecular mechanism through which cannabinoid molecules may directly impact the progression of this debilitating disease.
Keywords Cannabinoids; Alzheimer’s disease; Acetylcholinesterase
Introduction Since the characterization of the Cannabis sativa-produced cannabinoid, Δ9tetrahydrocannabinol (THC) (Figure 1), in the 1960’s,1 this natural product has been widely explored as an anti-emetic, anti-convulsive, anti-inflammatory, and analgesic.2 In these contexts, efficacy results from THC binding to the family of cannabinoid receptors found primarily on central and peripheral neurons (CB1) or immune cells (CB2).3 More recently, a link between the endocannabinoid system and Alzheimer’s disease has been discovered4 which has provided a new therapeutic target for the treatment of patients suffering from Alzheimer’s disease.5 New targets for this debilitating disease are critical as Alzheimer’s disease afflicts over 20 million people worldwide, with the number of diagnosed cases continuing to rise at
Correspondence to: Kim D. Janda. †Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, and Worm Institute for Research and Medicine (WIRM) ‡Department of Molecular Biology §Molecular and Integrated Neurosciences Department (MIND)
NIH Public Access Author Manuscript Mol Pharm. Author manuscript; available in PMC 2008 October 6.
Published in final edited form as: Mol Pharm. 2006 ; 3(6): 773–777. doi:10.1021/mp060066m.
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an exponential rate.6,7 These studies have demonstrated the ability of cannabinoids to provide neuroprotection against β-amyloid peptide (Aβ) toxicity.8-10 Yet, it is important to note that in these reports, cannabinoids serve as signaling molecules which regulate downstream events implicated in Alzheimer’s disease pathology and are not directly implicated as effecting Aβ at a molecular level.
One of the primary neuropathological hallmarks of Alzheimer’s disease is deposition of Aβ into amyloid plaques in areas of the brain important for memory and cognition.11 Over the last two decades, the etiology of Alzheimer’s disease has been elucidated through extensive biochemical and neurobiological studies, leading to an assortment of possible therapeutic strategies including prevention of downstream neurotoxic events, interference with Aβ metabolism, and reduction of damage from oxidative stress and inflammation.12 The impairment of the cholinergic system is the most dramatic of the neurotransmitter systems affected by Alzheimer’s disease and as a result, has been thoroughly investigated. Currently, there are four FDA-approved drugs that treat the symptoms of Alzheimer’s disease by inhibiting the active site of acetylcholinesterase (AChE), the enzyme responsible for the degradation of acetylcholine, thereby raising the levels of neurotransmitter in the synaptic cleft.13 In addition, AChE has been shown to play a further role in Alzheimer’s disease by acting as a molecular chaperone, accelerating the formation of amyloid fibrils in the brain and forming stable complexes with Aβ at a region known as the peripheral anionic binding site (PAS).14,15 Evidence supporting this theory was provided by studies demonstrating that the PAS ligand, propidium, is able to prevent amyloid acceleration in vitro, whereas active-site inhibitors had no effect.16 Due to the association between the AChE PAS and Alzheimer’s disease, a number of studies have focused on blocking this allosteric site.17 Recently, we reported a combined computational and experimental approach to identify compounds containing rigid, aromatic scaffolds hypothesized to disrupt protein-protein interactions.18-20 Similarly, THC is highly lipophilic in nature and possesses a fused tricyclic structure. Thus, we hypothesized that this terpenoid also could bind to the allosteric PAS of AChE with concomitant prevention of AChEpromoted Aβ aggregation.
Experimental Section Docking procedures THC was docked to the mouse AChE structure (PDB ID code 1J07) using AutoDock 220.127.116.11 Twenty docking runs (100 million energy evaluations each) were run with a 26.25 Å × 18.75 Å × 26.25 Å grid box with 0.375 Å grid spacing. This grid box was designed to include regions of both the catalytic site and the peripheral anionic site. Otherwise, standard docking settings were used for the AutoDock calculations, as previously detailed.18
Acetylcholinesterase inhibition studies All assays were performed using a Cary 50 Bio UV-visible spectrophotometer using an 18cell changer, and conducted at 37 °C, using a Cary PCB 150 Water Peltier System. Solutions of acetylthiocholine iodide (ATCh iodide) and 5,5′-dithio-bis-(2-nitrobenzoic) acid (DTNB) were prepared according to the method of Ellman, et al.22 Stock solutions of acetylcholinesterase from E. electricus were prepared by dissolving commercially available enzyme in 1% gelatin. Prior to use, an aliquot of the gelatin solution was diluted 1:200 in water. For the assay, the solution was diluted until enzyme activity between 0.10-0.13 AU/min at 500 μM ACTh iodide was obtained. Compounds were prepared as solutions in methanol. Assays were performed by mixing AChE, THC, and 340 μM DTNB in 100 mM phosphate buffer, pH 8.0, containing 5% methanol. Solutions were incubated at 37 °C for five minutes before the reaction was initiated by the addition of ATCh iodide (75 – 300 μM). The increase
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of absorbance at 412 nm was monitored for 2 to 5 min. All assays were run in triplicate. Initial rates were determined by subtracting the average observed initial rate from the non-enzymatic reaction.
Linear regression analysis of reciprocal plots of 1/νo versus 1/[S] for four THC concentrations was performed using Microsoft Excel software. The slope1/v was plotted against [I] to give Ki values. Propagation of error was performed to determine the error, ΔKi.
For studies to determine the mutual exclusivity of THC and propidium iodide, experiments were performed identically to simple THC inhibition studies with a fixed concentration of ACTh iodide (125 μM), and varied concentrations of propidium iodide (0-25 μM) and THC (0-15 μM).
AChE-induced β-amyloid peptide aggregation in the presence of AChE ligands The aggregation of the β-amyloid peptide was measured using the thioflavin T-based fluorometric assay as described by LeVine23 and Bartolini.16 Assays were measured using a SpectraMAX Gemini fluorescence plate reader with SOFTmax PRO 2.6.1 software. Aβ1-40 stock solutions were prepared in DMSO and HuAChE stocks prepared in distilled water. All stock solutions of Aβ and HuAChE were used immediately after preparation. In a 96-well plate, triplicate samples of a 20 μL solution of 23 nM of Aβ, 2.30 μM HuAChE and various concentrations of THC in 0.215 M sodium phosphate buffer, pH 8.0 were prepared. These solutions were incubated at room temperature along with triplicate solutions of Aβ alone, Aβ and AChE, and Aβ plus THC at various concentrations. After 48 h, a 2 μL aliquot was removed from each well, placed in a black-walled, clear-bottomed 96-well plate, and diluted with 50 mM glycine-NaOH buffer, pH 8.5, containing 1.5 μM thioflavin T to a total volume of 200 μL. After incubating for 5 min, the fluorescence was measured using λexc = 466 nm and λem = 490 nm with excitation and emission slits of 2 nm. The fluorescence emission spectrum was recorded between 450 and 600 nm, with excitation at 446 nm.
The fluorescence intensities were averaged, and the average background fluorescence of buffer only, or buffer and THC, was subtracted. The corrected fluorescence values were plotted with their standard deviation. The equation, Fi/Fo × 100%, where Fi is the fluorescence of AChE, Aβ, and THC, and Fo is the fluorescence of AChE and Aβ, was used to quantify the extent to which each compound inhibits Aβ aggregation. The student’s t-test function of Microsoft Excel was used to determine p values and assess statistical significance between reactions.
Control experiments containing AChE, THC, and thioflavin T or AChE and thioflavin T alone were also performed to ensure that any observed fluorescence decrease was not attributable to the molecular rotor properties of thioflavin T upon binding to AChE. For these reactions, all concentrations were identical to those used in the described Aβ aggregation assays (vide supra).
Results and Discussion THC binding to AChE initially was modeled in silico using AutoDock 18.104.22.168 Twenty docking runs with 100 million energy evaluations each were performed with a 26.25 Å × 18.75 Å × 26.25 Å grid box with 0.375 Å grid spacing, which included regions of both the catalytic site and the PAS. Examination of the docking results revealed that THC was predicted to bind to AChE with comparable affinity to the best reported PAS binders, with the primary binding interaction observed between the ABC fused ring of the THC scaffold and the Trp86 indole side chain of AChE (Figure 2). Further interactions were also evident between THC and the backbone carbonyls of Phe123 and Ser125. Encouraged by these results, we tested the ability
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of THC to inhibit AChE catalytic activity. Steady-state kinetic analysis of THC inhibition revealed that THC competitively inhibits AChE (Ki = 10.2 μM) (Figure 3A). This level of inhibition is relatively modest, yet it is important to note that inhibition of acetylcholine cleavage is not a prerequisite for effective reduction of Aβ aggregation; indeed, most PAS binders are moderate AChE inhibitors displaying either non-competitive or mixed-type inhibition.16 While THC shows competitive inhibition relative to the substrate, this does not necessitate a direct interaction between THC and the AChE active site. In fact, given the proximity of the PAS to the protein channel leading to the catalytic triad active site, it is possible to block substrate entry into the active site while bound to the PAS, thus preventing the formation of an ESI complex.18,24 In order to test this hypothesis, additional kinetic experiments were performed to determine the mutual exclusivity of THC and propidium, a well characterized purely noncompetitive AChE inhibitor and PAS binder. Dixon plots of ν-1 versus propidium concentration at varying concentrations of THC returned a series of parallel lines, indicating that THC and propidium cannot bind simultaneously to AChE (Figure 3B). Thus, these studies verify our docking results and demonstrate that THC and propidium are mutually exclusive PAS inhibitors. Additionally, recent reports have suggested that the selectivity of a given inhibitor for AChE over butyrylcholinesterase (BuChE) can be correlated with the ability of a compound to block AChE-accelerated Aβ aggregation.25,26 Kinetic examination of BuChE inhibition revealed a slight reduction in enzymatic activity at high concentrations of THC (IC50 ≥ 100 μM); however, these experiments were limited by the poor solubility of THC in aqueous solution.
The activity of THC towards the inhibition of Aβ aggregation was then investigated using a thioflavin T (ThT)-based fluorometric assay to stain putative Aβ fibrils.23 Using this assay, we found that THC is an effective inhibitor of the amyloidogenic effect of AChE (Figure 4). In fact, at a concentration of 50 μM, propidium does not fully prevent AChE-induced aggregation (p = 0.03, student’s T-test), while THC completely blocks the AChE effect on Aβ aggregation, with significantly greater inhibition than propidium (p = 0.04, student’s Ttest), one of the most effective aggregation inhibitors reported to date.16 However, the observed decrease in fluorescence could also be rationalized as a result of a competition between THC and ThT for the same site on AChE. It has been shown that ThT also can bind to the PAS and that this binding leads to an increase in fluorescence. Presumably, this phenomenon results from ThT serving as a molecular rotor in which fluorescence quantum yield is sensitive to the intrinsic rotational relaxation; thus, when molecular rotation is slowed by protein binding, the quantum yield of the molecule can increase dramatically.27,28 In order to ensure that the observed fluorescence decrease was due to fibril inhibition, control experiments were performed using AChE, THC, and ThT. Reactions containing AChE and ThT alone showed the same fluorescence output as those containing AChE, THC, and ThT, providing convincing evidence that any observed reduction in fluorescence can be attributed to fewer Aβ fibrils.
Conclusion We have demonstrated that THC competitively inhibits AChE, and furthermore, binds to the AChE PAS and diminishes Aβ aggregation. In contrast to previous studies aimed at utilizing cannabinoids in Alzheimer’s disease therapy,8-10 our results provide a mechanism whereby the THC molecule can directly impact Alzheimer’s disease pathology. We note that while THC provides an interesting Alzheimer’s disease drug lead, it is a psychoactive compound with strong affinity for endogenous cannabinoid receptors. It is noteworthy that THC is a considerably more effective inhibitor of AChE-induced Aβ deposition than the approved drugs for Alzheimer’s disease treatment, donepezil and tacrine, which reduced Aβ aggregation by only 22% and 7%, respectively, at twice the concentration used in our studies.7 Therefore, AChE inhibitors such as THC and its analogues may provide an improved therapeutic for Alzheimer’s disease, augmenting acetylcholine levels by preventing neurotransmitter
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degradation and reducing Aβ aggregation, thereby simultaneously treating both the symptoms and progression of Alzheimer’s disease.
This work was supported by the Skaggs Institute for Chemical Biology and a NIH Kirschstein National Research Service Award to L.M.E.
Marijuana’s Active Ingredient Shown to Inhibit Primary Marker of Alzheimer’s Disease
Discovery Could Lead to More Effective Treatments
LA JOLLA, CA, August 9, 2006 – Scientists at The Scripps Research Institute have found that the active ingredient in marijuana, tetrahydrocannabinol or THC, inhibits the formation of amyloid plaque, the primary pathological marker for Alzheimer’s disease. In fact, the study said, THC is “a considerably superior inhibitor of [amyloid plaque] aggregation” to several currently approved drugs for treating the disease.
The study was published online August 9 in the journal Molecular Pharmaceutics, a publication of the American Chemical Society.
According to the new Scripps Research study, which used both computer modeling and biochemical assays, THC inhibits the enzyme acetylcholinesterase (AChE), which acts as a “molecular chaperone” to accelerate the formation of amyloid plaque in the brains of Alzheimer victims. Although experts disagree on whether the presence of beta-amyloid plaques in those areas critical to memory and cognition is a symptom or cause, it remains a significant hallmark of the disease. With its strong inhibitory abilities, the study said, THC “may provide an improved therapeutic for Alzheimer’s disease” that would treat “both the symptoms and progression” of the disease.
“While we are certainly not advocating the use of illegal drugs, these findings offer convincing evidence that THC possesses remarkable inhibitory qualities, especially when compared to AChE inhibitors currently available to patients,” said Kim Janda, Ph.D., who is Ely R. Callaway, Jr. Professor of Chemistry at Scripps Research, a member of The Skaggs Institute for Chemical Biology, and director of the Worm Institute of Research and Medicine. “In a test against propidium, one of the most effective inhibitors reported to date, THC blocked AChE-induced aggregation completely, while the propidium did not. Although our study is far from final, it does show that there is a previously unrecognized molecular mechanism through which THC may directly affect the progression of Alzheimer’s disease.”
As the new study points out, any new treatment that could halt or even slow the progression of Alzheimer’s disease would have a major impact on the quality of life for patients, as well as reducing the staggering health care costs associated with the disease.
Alzheimer’s disease is the leading cause of dementia among the elderly, and the numbers are growing. The Alzheimer’s Association estimates 4.5 million Americans have the disease, a figure that could reach as high as 16 million by 2050. A survey by the National Center for Health Statistics noted that half of all nursing home residents have Alzheimer’s disease or a related disorder. The costs of caring for Alzheimer’s patients are at least $100 billion annually, according to the National Institute on Aging.
Over the last two decades, the causes of Alzheimer’s disease have been clarified through extensive biochemical and neurobiological studies, leading to an assortment of possible therapeutic strategies including interference with beta amyloid metabolism, the focus of the Scripps Research study.
The cholinergic system – the nerve cell system in the brain that uses acetylcholine (Ach) as a neurotransmitter – is the most dramatic of the neurotransmitter systems affected by Alzheimer’s disease. Levels of acetylcholine, which was first identified in 1914, are abnormally low in the brains of Alzheimer’s patients. Currently, there are four FDA-approved drugs that treat the symptoms of Alzheimer’s disease by inhibiting the active site of acetylcholinesterase, the enzyme responsible for the degradation of acetylcholine.
“When we investigated the power of THC to inhibit the aggregation of beta-amyloid,” Janda said, “we found that THC was a very effective inhibitor of acetylcholinesterase. In addition to propidium, we also found that THC was considerably more effective than two of the approved drugs for Alzheimer’s disease treatment, donepezil (Aricept ®) and tacrine (Cognex ®), which reduced amyloid aggregation by only 22 percent and 7 percent, respectively, at twice the concentration used in our studies. Our results are conclusive enough to warrant further investigation.”
Other authors of the study, titled “A Molecular Link Between the Active Component of Marijuana and Alzheimer’s Disease Pathology,” include Lisa M. Eubanks, Claude J. Rogers, and Tobin J. Dickerson of The Scripps Research Institute, the Skaggs Institute for Chemical Biology, and the Worm Institute for Research and Medicine; and Albert E. Beuscher IV, George F. Koob, and Arthur J. Olson of The Scripps Research Institute.
The study was supported by the Skaggs Institute for Chemical Biology at Scripps Research and the National Institutes of Health.
About The Scripps Research Institute
The Scripps Research Institute is one of the world’s largest independent, non-profit biomedical research organizations, at the forefront of basic biomedical science that seeks to comprehend the most fundamental processes of life. Scripps Research is internationally recognized for its discoveries in immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune, cardiovascular, and infectious diseases, and synthetic vaccine development. Established in its current configuration in 1961, it employs approximately 3,000 scientists, postdoctoral fellows, scientific and other technicians, doctoral degree graduate students, and administrative and technical support personnel. Scripps Research is headquartered in La Jolla, California. It also includes Scripps Florida, whose researchers focus on basic biomedical science, drug discovery, and technology development. Currently operating from temporary facilities in Jupiter, Scripps Florida will move to its permanent campus in 2009.
Cannabinol delays symptom onset in SOD1 (G93A) transgenic mice without affecting survival.
- Department of Neurology, University of Washington, Seattle, WA 98195, USA. firstname.lastname@example.org
Therapeutic options for amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disorder, remain limited. Emerging evidence from clinical studies and transgenic mouse models of ALS suggests that cannabinoids, the bioactive ingredients of marijuana (Cannabis sativa) might have some therapeutic benefit in this disease. However, Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the predominant cannabinoid in marijuana, induces mind-altering effects and is partially addictive, compromising its clinical usefulness. We therefore tested whether cannabinol (CBN), a non-psychotropic cannabinoid, influences disease progression and survival in the SOD1 (G93A) mouse model of ALS. CBN was delivered via subcutaneously implanted osmotic mini-pumps (5 mg/kg/day) over a period of up to 12 weeks. We found that this treatment significantly delays disease onset by more than two weeks while survival was not affected. Further research is necessary to determine whether non-psychotropic cannabinoids might be useful in ameliorating symptoms in ALS.