Therapeutic properties of multi-cannabinoid treatment strategies for Alzheimer's disease.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease characterized by declining cognition and behavioral impairment, and hallmarked by extracellular amyloid-ß plaques, intracellular neurofibrillary tangles (NFT), oxidative stress, neuroinflammation, and neurodegeneration. There is currently no cure for AD and approved treatments do not halt or slow disease progression, highlighting the need for novel therapeutic strategies. Importantly, the endocannabinoid system (ECS) is affected in AD. Phytocannabinoids, including cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), interact with the ECS, have anti-inflammatory, antioxidant, and neuroprotective properties, can ameliorate amyloid-ß and NFT-related pathologies, and promote neurogenesis. Thus, in recent years, purified CBD and THC have been evaluated for their therapeutic potential. CBD reversed and prevented the development of cognitive deficits in AD rodent models, and low-dose THC improved cognition in aging mice. Importantly, CBD, THC, and other phytochemicals present in Cannabis sativa interact with each other in a synergistic fashion (the "entourage effect") and have greater therapeutic potential when administered together, rather than individually. Thus, treatment of AD using a multi-cannabinoid strategy (such as whole plant cannabis extracts or particular CBD:THC combinations) may be more efficacious compared to cannabinoid isolate treatment strategies. Here, we review the current evidence for the validity of using multi-cannabinoid formulations for AD therapy. We discuss that such treatment strategies appear valid for AD therapy but further investigations, particularly clinical studies, are required to determine optimal dose and ratio of cannabinoids for superior effectiveness and limiting potential side effects. Furthermore, it is pertinent that future in vivo and clinical investigations consider sex effects.

CuATSM improves motor function and extends survival but is not tolerated at a high dose in SOD1G93A mice with a C57BL/6 background.

The synthetic copper-containing compound, CuATSM, has emerged as one of the most promising drug candidates developed for the treatment of amyotrophic lateral sclerosis (ALS). Multiple studies have reported CuATSM treatment provides therapeutic efficacy in various mouse models of ALS without any observable adverse effects. Moreover, recent results from an open label clinical study suggested that daily oral dosing with CuATSM slows disease progression in patients with both sporadic and familial ALS, providing encouraging support for CuATSM in the treatment of ALS. Here, we assessed CuATSM in high copy SOD1G93A mice on the congenic C57BL/6 background, treating at 100 mg/kg/day by gavage, starting at 70 days of age. This dose in this specific model has not been assessed previously. Unexpectedly, we report a subset of mice initially administered CuATSM exhibited signs of clinical toxicity, that necessitated euthanasia in extremis after 3-51 days of treatment. Following a 1-week washout period, the remaining mice resumed treatment at the reduced dose of 60 mg/kg/day. At this revised dose, treatment with CuATSM slowed disease progression and increased survival relative to vehicle-treated littermates. This work provides the first evidence that CuATSM produces positive disease-modifying outcomes in high copy SOD1G93A mice on a congenic C57BL/6 background. Furthermore, results from the 100 mg/kg/day phase of the study support dose escalation determination of tolerability as a prudent step when assessing treatments in previously unassessed models or genetic backgrounds.

Chronic cannabidiol (CBD) treatment did not exhibit beneficial effects in 4-month-old male TAU58/2 transgenic mice.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline, motor impairments, and accumulation of hallmark proteins, amyloid-beta (Aß) and tau. Traditionally, transgenic mouse models for AD have focused on Aß pathology, however, recently a number of tauopathy transgenic models have been developed, including the TAU58/2 transgenic model. Cannabidiol (CBD), a non-toxic constituent of the Cannabis sativa plant, has been shown to prevent and reverse cognitive deficits in Aß transgenic mouse models of AD. Importantly, the therapeutic properties of CBD on the behavioural phenotype of tauopathy mouse models have not been investigated. We assessed the impact of chronic CBD treatment (i.e. 50 mg/kg CBD i.p. administration starting 3 weeks prior to behavioural assessments) on disease-relevant behaviours of 4-month-old TAU58/2 transgenic males in paradigms for anxiety, motor functions, and cognition. TAU58/2 transgenic males demonstrated reduced body weight, anxiety and impaired motor functions. Furthermore, they demonstrated increased freezing in fear conditioning compared to wild type-like animals. Interestingly, both sociability and social recognition memory were intact in AD transgenic mice. Chronic CBD treatment did not affect behavioural changes in transgenic males. In summary, 4-month-old TAU58/2 transgenic males exhibited no deficits in social recognition memory, suggesting that motor deficits and changes in anxiety at this age do not impact on social domains. The moderate increase in fear-associated memory needs further investigation but could be related to differences in fear extinction. Future investigations will need to clarify CBD's therapeutic potential for reversing motor deficits in TAU58/2 transgenic mice by considering alternative CBD treatment designs including changed CBD dosing.

Cannabidiol (CBD) reduces anxiety-related behavior in mice via an FMRP-independent mechanism.

Fragile X Syndrome is a neurodevelopmental disorder which affects intellectual, social and physical development due to mutation of the Fragile X mental retardation 1 (FMR1) gene. The resultant loss of Fragile X mental retardation protein can be modelled by Fmr1 gene knockout (KO) in mice. The current study investigated the behavioural effects of cannabidiol (CBD; a non-psychoactive phytocannabinoid) in male Fmr1 KO mice as a preclinical model for therapeutic discovery. Vehicle or CBD (5 or 20 mg/kg body weight) was administered to adult Fmr1 KO and wild type-like (WT) mice before they were tested in behavioural tasks including: open field (OF), elevated plus maze (EPM), spontaneous alternation, social preference, and passive avoidance tasks. Fmr1 KO mice were hyperlocomotive and hyperexplorative and habituated more slowly to a novel environment compared to control animals. Furthermore, Fmr1 KO mice showed fewer anxiety-related behaviours across tests. Effects of CBD were subtle and limited to the EPM, where CBD decreased the anxiety response of all mice tested. Acute CBD had no impact on locomotion or anxiety-related parameters in the OF. Cognitive performance of Fmr1 KO mice was equivalent to controls and not affected by CBD treatment. Brain concentrations of CBD were equivalent between genotypes, but in animals sacrificed 90 min post-administration, decreased plasma CBD in Fmr1 KO mice compared to WT suggested more rapid clearance of CBD by transgenic animals. Overall, acute CBD at the doses chosen did not selectively normalize behavioural abnormalities in Fmr1 KO mice, but reduced anxiety-like behaviour in both Fmr1 KO and WT mice.

Targeting Inflammatory Pathways in Alzheimer's Disease: A Focus on Natural Products and Phytomedicines.

Studies of the brains of Alzheimer's disease (AD) patients have revealed key neuropathological features, such as the deposition of aggregates of insoluble amyloid-ß (Aß) peptides and neurofibrillary tangles (NFTs). These pathological protein deposits, including Aß peptides (which form senile plaques) and hyperphosphorylated tau (which aggregates into NFTs), have been assumed to be 'the cause of AD'. Aß has been extensively targeted to develop an effective disease-modifying therapy, but with limited clinical success. Emerging therapies are also now targeting further pathological processes in AD, including neuroinflammation. This review focuses on the inflammatory and oxidative stress-related changes that occur in AD, and discusses some emerging anti-inflammatory natural products and phytomedicines. Many of the promising compounds are cytokine-suppressive anti-inflammatory drugs (CSAIDs), which target the proinflammatory AP1 and nuclear factor-κB signalling pathways and inhibit the expression of many proinflammatory cytokines, such as interleukin (IL)-1, IL-6, tumour necrosis factor-α, or nitric oxide produced by inducible nitric oxide synthase. However, many of these phytomedicines have not been tested in rigorous clinical trials in AD patients. It is not yet clear if the active compounds reach an effective concentration in the brain (due to limited bioavailability) or if they can slow down AD progression in long-term trials. The authors suggest that it is crucial for both the pharmacological and complementary medicine industries to conduct and fund those studies to significantly advance the field.

Assessment of diets containing curcumin, epigallocatechin-3-gallate, docosahexaenoic acid and α-lipoic acid on amyloid load and inflammation in a male transgenic mouse model of Alzheimer's disease: Are combinations more effective?

Increasingly, evidence is accumulating pointing at a protective role of a healthy diet at decreasing the risk of Alzheimer's disease. To test the effectiveness of nutritional components, the following food-derived compounds: curcumin alone (curcumin), curcumin combined with (-)epigallocatechin-3-gallate (EGCG), docosahexaenoic acid (DHA) and α-lipoic acid (ALA) (curcumin + EDA), or a combination of EGCG, DHA and ALA (EDA) were assessed in male Tg2576 transgenic mice on amyloid plaque load, amyloid levels (Aß40/Aß42, but not oligomers due to tissue limitations), microglial activation and memory using the contextual and cued fear conditioning test. The combination diet EDA, resulted in the strongest reduction of amyloid plaque load in both the cortical (p < .0001) and hippocampal (p < .0001) areas of the Tg2576 mouse brain, along with lower Aß40/Aß42 levels in the frontal cortex (p = .000129 and p = .000039, respectively) and Aß42 levels in the temporal lobe (p = .000082). A curcumin only diet was shown to lower amyloid plaque load (p = .028), but when combined with EGCG, DHA and ALA did not result in further decreases in amyloid plaque load. The EDA combination group showed the most prominent decrease in microglial activation (number of microglia around plaques: p < .05 and p < .0001, respectively, for the cortex and hippocampus). Analysing the hippocampal associated contextual fear conditioning revealed that both the curcumin+EDA (p < .0001) and EDA groups (p = .001) spent increased time on freezing compared to the control group. In addition, the curcumin+EDA group showed a significant increase in time spent freezing compared with the curcumin only group. In the amygdala associated cued test, all mice demonstrated the ability to associate the conditioned stimulus with the unconditioned stimulus as evidenced by a significant increase in freezing behaviour in response to the presentation of the cue (p < .0001). Post-hoc analysis showed that only curcumin+EDA (p < .0001) and EDA groups (p < .0001) developed a significant increase in freezing during the cue presentation. The results from this study show that the combination of EGCG, DHA and ALA (EDA) appeared to have the most potent anti-inflammatory and neuroprotective effect. Our results also demonstrate that interactions between nutraceutical products might result in counterproductive outcomes, highlighting the fact that manufacturers of nutraceuticals containing multiple compounds should be careful not to claim additive or synergistic effects of their combination products in vivo without having tested it in animal models and/or human clinical trials.

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-ß 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.

Novel behavioural characteristics of the APP(Swe)/PS1ΔE9 transgenic mouse model of Alzheimer's disease.

In order to better understand animal models of Alzheimer's disease, novel phenotyping strategies have been established for transgenic mouse models. In line with this, the current study characterised male APPxPS1 transgenic mice on mixed C57BL/6JxC3H/HeJ background for the first time for social recognition memory, sensorimotor gating, and spatial memory using the cheeseboard test as an alternative to the Morris water maze. Furthermore, locomotion, anxiety, and fear conditioning were evaluated in transgenic and wild type-like animals. APPxPS1 males displayed task-dependent hyperlocomotion and anxiety behaviours and exhibited social recognition memory impairments compared to wild type-like littermates. Spatial learning and memory, fear conditioning, and sensorimotor gating were unaffected in APPxPS1 transgenic mice. In conclusion, this study describes for the first time social recognition memory deficits in male APPxPS1 mice and suggests that spatial learning and memory deficits reported in earlier studies are dependent on the sex and genetic background of the APPxPS1 mouse line used. Furthermore, particular test conditions of anxiety and spatial memory paradigms appear to impact on the behavioural response of this transgenic mouse model for Alzheimer's disease.

Neuregulin 1 hypomorphic mutant mice: enhanced baseline locomotor activity but normal psychotropic drug-induced hyperlocomotion and prepulse inhibition regulation.

Neuregulin 1 (Nrg1) has been widely recognized as a candidate gene for schizophrenia. This study therefore investigated mice heterozygous for a mutation in the transmembrane domain of this trophic factor (Nrg1+/- mice) in a number of behavioural test systems with relevance to schizophrenia, including psychotropic drug-induced locomotor hyperactivity and prepulse inhibition (PPI) of startle. Baseline locomotor activity in the open field or in photocell cages was slightly, but significantly enhanced in Nrg1+/- mice compared to wild-type littermate controls at age 12-16 wk, but not at age 6 months. The ability of amphetamine, phencyclidine (PCP) or MK-801 to induce locomotor hyperactivity was not significantly different between the genotypes. There was no difference in baseline PPI, startle or startle habituation and there was no difference in the effect of apomorphine, amphetamine or MK-801 on any of these parameters. Only treatment with the 5-HT1A receptor agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) showed a differential effect between genotypes, with a disruption of PPI occurring in Nrg1+/- mice compared to no effect in wild-type controls. This treatment also induced a significant reduction of startle which could have influenced the result. The density of dopamine D2 receptors in the forebrain and of 5-HT1A receptors in the hippocampus and raphe nuclei was not different between Nrg1+/- mice and controls. These studies add to the knowledge about behavioural effects in this mouse model of impaired Nrg1 function and suggest that a number of the behavioural tests with relevance to schizophrenia are normal in these mice.

Y2Y4 receptor double knockout protects against obesity due to a high-fat diet or Y1 receptor deficiency in mice.

Neuropeptide Y receptors are critical regulators of energy homeostasis, but the functional interactions and relative contributions of Y receptors and the environment in this process are unknown. We measured the effects of an ad libitum diet of normal or high-fat food on energy balance in mice with single, double, or triple deficiencies of Y1, Y2, or Y4 receptors. Whereas wild-type mice developed diet-induced obesity, Y2Y4 double knockouts did not. In contrast, Y1 knockout or Y1Y2 or Y1Y4 receptor double knockout mice developed an exacerbated diet-induced obesity syndrome. Remarkably, the antiobesity effect of Y2Y4 deficiency was stronger than the obesogenic effect of Y1 deficiency, since Y1Y2Y4 triple knockouts did not develop obesity on the high-fat diet. Resistance to diet-induced obesity in Y2Y4 knockouts was associated with reduced food intake and improved glucose tolerance in the absence of changes in total physical activity. Fecal concentration of free fatty acids was significantly increased in Y2Y4 knockouts in association with a significantly reduced bile acid pool and marked alterations in intestinal morphology. In addition, hypothalamic proopiomelanocortin expression was decreased in diet-induced obesity (in both wild-type and Y1 receptor knockout mice) but not in obesity-resistant Y2Y4 receptor knockout mice fed a high-fat diet. Therefore, deletion of Y2 and Y4 receptors synergistically protects against diet-induced obesity, at least partially via changes in food intake and hypothalamic proopiomelanocortin expression.

Behavioral phenotyping of mice in pharmacological and toxicological research.

The evaluation of behavioral effects is an important component for the in vivo screening of drugs or potentially toxic compounds in mice. Ideally, such screening should be composed of monitoring general health, sensory functions, and motor abilities, right before specific behavioral domains are tested. A rational strategy in the design and procedure of testing as well as an effective composition of different well-established and reproducible behavioral tests can minimize the risk of false positive and false negative results in drug screening. In the present review we describe such basic considerations in planning experiments, selecting strains of mice, and propose groups of behavioral tasks suitable for a reliable detection of differences in specific behavioral domains in mice. Screening of general health and neurophysiologic functions (reflexes, sensory abilities) and motor function (pole test, wire hang test, beam walking, rotarod, accelerod, and footprint) as well as specific hypothesis-guided testing in the behavioral domains of learning and memory (water maze, radial maze, conditioned fear, and avoidance tasks), emotionality (open field, hole board, elevated plus maze, and object exploration), nociception (tail flick, hot plate), psychiatric-like conditions (porsolt swim test, acoustic startle response, and prepulse inhibition), and aggression (isolation-induced aggression, spontaneous aggression, and territorial aggression) are described in further detail. This review is designed to describe a general approach, which increases reliability of behavioral screening. Furthermore, it provides an overview on a selection of specific procedures suitable for but not limited to behavioral screening in pharmacology and toxicology.