Chronic nSMase inhibition suppresses neuronal exosome spreading and sex-specifically attenuates amyloid pathology in APP knock-in Alzheimer's disease mice.

Female biased pathology and cognitive decline in Alzheimer's disease (AD) have been consistently observed with unclear underlying mechanisms. Although brain sphingolipid ceramide is elevated in AD patients, whether and how ceramide may contribute to sex-specific differences in amyloid pathology is unknown. Here we investigated the sex-specific impact of chronic pharmacological inhibition of neutral sphingomyelinase (nSMase), a key enzyme responsible for ceramide metabolism, on in vivo neuron-derived exosome dynamics, Aß plaque load, and cognitive function in the APPNL-F/NL-F knock-in (APP NL-F) AD mouse model. Our results found sex-specific increase of cortical C20:0 ceramide and brain exosome levels only in APP NL-F but not in age-matched WT mice. Although nSMase inhibition similarly blocks exosome spreading in male and female mice, significantly reduced amyloid pathology was mostly observed in cortex and hippocampus of female APP NL-F mice with only modest effect found on male APP NL-F mice. Consistently, T maze test to examine spatial working memory revealed a female-specific reduction in spontaneous alternation rate in APP NL-F mice, which was fully reversed with chronic nSMase inhibition. Together, our results suggest that disease induced changes in ceramide and exosome pathways contribute to the progression of female-specific amyloid pathology in APP NL-F AD models.

Targeting the CB2 receptor and other endocannabinoid elements to delay disease progression in amyotrophic lateral sclerosis.

Cannabinoids form a singular group of plant-derived compounds, endogenous lipids and synthetic derivatives with multiple therapeutic effects exerted by targeting different elements of the endocannabinoid system. One of their therapeutic applications is the preservation of neuronal integrity exerted by attenuating the multiple neurotoxic events that kill neurons in neurodegenerative disorders. In this review, we will address the potential of cannabinoids as neuroprotective agents in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disorder characterized by muscle denervation, atrophy and paralysis, and progressive deterioration in upper and/or lower motor neurons. The emphasis will be paid on the cannabinoid type 2 (CB2 ) receptor, whose activation limits glial reactivity, but the potential of additional endocannabinoid-related targets will be also addressed. The evidence accumulated so far at the preclinical level supports the need to soon move towards the patients and initiate clinical trials to confirm the potential of cannabinoid-based medicines as disease modifiers in ALS. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.6/issuetoc.

Elevated exosomal secretion of miR-124-3p from spinal neurons positively associates with disease severity in ALS.

MicroRNAs (miRs) are powerful regulators of CNS development and diseases. Plasma and cerebrospinal fluid (CSF) miRs have recently been implicated as potential new sources for biomarker development. Previously we showed that miR-124-3p, an essential miR for neuronal identity, is highly abundant in neuronal exosomes and its expression decreases in spinal cord of ALS model SOD1G93A mice. In the current study, we found a disease associated reduction of miR-124-3p levels specifically in spinal neurons using in situ hybridization. By employing our recently developed exosome reporter mice in combination with sciatic nerve injections, we observed an increased association of miR-124-3p with spinal motor neuron-derived exosomes in SOD1G93A mice, even at the pre-symptomatic stage. Sciatic nerve injection delivered miR-124-3p is also more frequently localized outside of spinal motor neurons in SOD1G93A mice. Subsequent quantitative analysis of miR-124-3p levels in CSF exosomes from ALS patients found a significant correlation between CSF exosomal miR-124-3p levels and disease stage (indicated by the ALSFRS-R score) of (male) ALS patients. These results provide preliminary evidence to support the potential use of CSF exosomal miR-124-3p as a disease stage indicator in ALS.

Pharmacokinetics of Sativex® in Dogs: Towards a Potential Cannabinoid-Based Therapy for Canine Disorders.

The phytocannabinoid-based medicine Sativex® is currently marketed for the treatment of spasticity and pain in multiple sclerosis patients and is being investigated for other central and peripheral pathological conditions. It may also serve in Veterinary Medicine for the treatment of domestic animals, in particular for dogs affected by different pathologies, including human-like pathological conditions. With the purpose of assessing different dosing paradigms for using Sativex in Veterinary Medicine, we investigated its pharmacokinetics when administered to naïve dogs via sublingual delivery. In the single dose arm of the study, adult Beagle dogs were treated with 3 consecutive sprays of Sativex, and blood samples were collected at 12 intervals up to 24 h later. In the multiple dose arm of the study, Beagle dogs received 3 sprays daily for 14 days, and blood samples were collected for 24 h post final dose. Blood was used to obtain plasma samples and to determine the levels of cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC) and its metabolite 11-hydroxy-Δ9-THC. Maximal plasma concentrations of both Δ9-THC (Cmax = 18.5 ng/mL) and CBD (Cmax = 10.5 ng/mL) were achieved 2 h after administration in the single dose condition and at 1 h in the multiple dose treatment (Δ9-THC: Cmax = 24.5 ng/mL; CBD: Cmax = 15.2 ng/mL). 11hydroxy-Δ9-THC, which is mainly formed in the liver from Δ9-THC, was almost undetected, which is consistent with the use of sublingual delivery. A potential progressive accumulation of both CBD and Δ9-THC was detected following repeated exposure, with maximum plasma concentrations for both cannabinoids being achieved following multiple dose. Neurological status, body temperature, respiratory rate and some hemodynamic parameters were also recorded in both conditions, but in general, no changes were observed. In conclusion, this study demonstrates that single or multiple dose sublingual administration of Sativex to naïve dogs results in the expected pharmacokinetic profile, with maximal levels of phytocannabinoids detected at 1-2 h and suggested progressive accumulation after the multiple dose treatment.

Targeting glial cannabinoid CB2 receptors to delay the progression of the pathological phenotype in TDP-43 (A315T) transgenic mice, a model of amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Cannabinoid CB2 receptors are up-regulated in reactive microglia in the spinal cord of TDP-43 (A315T) transgenic mice, an experimental model of amyotrophic lateral sclerosis. To determine whether this up-regulation can be exploited pharmacologically, we investigated the effects of different treatments that affect CB2 receptor function. EXPERIMENTAL APPROACH: We treated TDP-43 (A315T) transgenic mice with the non-selective agonist WIN55,212-2, alone or combined with selective CB1 or CB2 antagonists, as well as with the selective CB2 agonist HU-308, and evaluated their effects on the pathological phenotype. KEY RESULTS: WIN55,212-2 had modest beneficial effects in the rotarod test, Nissl staining of motor neurons, and GFAP and Iba-1 immunostainings in the spinal cord, which were mediated in part by CB2 receptor activation. HU-308 significantly improved the rotarod performance of the transgenic mice, with complete preservation of Nissl-stained motor neurons in the ventral horn. Reactive astrogliosis labelled with GFAP was also attenuated by HU-308 in the dorsal and ventral horns, in which CB2 receptors colocalize with this astroglial marker. Furthermore, HU-308 reduced the elevated Iba-1 immunostaining in the ventral horn of TDP-43 transgenic mice, but did not affect this immunoreactivity in white matter, in which CB2 receptors also colocalize with this microglial marker. CONCLUSIONS AND IMPLICATIONS: Our study shows an important role for glial CB2 receptors in limiting the progression of the pathological phenotype in TDP-43 (A315T) transgenic mice. Such benefits appear to derive from the activation of CB2 receptors concentrated in astrocytes and reactive microglia located in spinal dorsal and ventral horns. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Neuroprotective effects of the cannabigerol quinone derivative VCE-003.2 in SOD1G93A transgenic mice, an experimental model of amyotrophic lateral sclerosis.

Antioxidant phytocannabinoids, synthetic compounds targeting the CB2 receptor, and inhibitors of the endocannabinoid inactivation afforded neuroprotection in SOD1G93A mutant mice, a model of ALS. These effects may involve the activation of PPAR-γ too. Here, we have investigated the neuroprotective effects in SOD1G93A mutant mice of the cannabigerol derivative VCE-003.2, which works as neuroprotectant by activating PPAR-γ. Mice were treated with VCE-003.2 from 60 days up to an advanced stage in disease progression (18 weeks), when they were euthanized and used for analysis of neuropathological signs. As expected, SOD1G93A transgenic mice experienced a progressive weight loss and neurological deterioration, which was associated with a marked loss of spinal cholinergic motor neurons, glial reactivity, and elevations in several biochemical markers (cytokines, glutamate transporters) that indirectly reflect the glial proliferation and activation in the spinal cord. The treatment with VCE-003.2 improved most of these neuropathological signs. It attenuated the weight loss and the anomalies in neurological parameters, preserved spinal cholinergic motor neurons, and reduced astroglial reactivity. VCE-003.2 also reduced the elevations in IL-1ß and glial glutamate transporters. Lastly, VCE-003.2 attenuated the LPS-induced generation of TNF-α and IL-1ß in cultured astrocytes obtained from SOD1G93A transgenic newborns, an effect also produced by rosiglitazone, then indicating a probable PPAR-γ activation as responsible of its neuroprotective effects. In summary, our results showed benefits with VCE-003.2 in SOD1G93A transgenic mice supporting PPAR-γ as an additional neuroprotective target available for cannabinoids in ALS. Such benefits would need to be validated in other ALS models prior to be translated to the clinical level.

Analysis of endocannabinoid receptors and enzymes in the post-mortem motor cortex and spinal cord of amyotrophic lateral sclerosis patients.

OBJECTIVE: We have investigated the endocannabinoid system in the motor cortex of motor neuron disease (MND) patients. METHODS: Post-mortem samples from MND patients and controls were used for immunostaining and/or Western blotting analysis of endocannabinoid elements. RESULTS: We did not find any evidence of neuronal losses in the motor cortex of MND patients, but elevations in glial markers Iba-1 and GFAP were evident. We found no changes in FAAH and MAGL enzymes and in the CB1 receptor, which correlated with the lack of cortical neuron death. By contrast, the Western blotting analysis of CB2 receptors proved an increase in the motor cortex corroborated by immunostaining, correlating with the elevated gliosis in these patients. Double-labeling analyses revealed that this elevated CB2 receptor immunostaining was located in GFAP-labelled astroglial cells. However, we also found CB2 receptor labeling in cortical neurons confirmed with double immunofluorescence with the neuronal marker MAP-2. This was also found in the spinal cord, using double-labeling with the spinal motor neuron marker choline-acetyl transferase. This happened in both patients and controls, despite these neurons experienced an important degeneration in patients reflected in reduced Nissl staining, TDP-43 immunostaining and CB1 receptor levels measured by Western blotting. CONCLUSION: We have confirmed that CB2 receptors are elevated in the motor cortex of MND patients associated with the reactive gliosis. This phenomenon is previous to neuronal losses. We also found CB2 receptors in cortical and spinal motor neurons. These observations support that targeting this receptor may serve for developing neuroprotective therapies in MNDs.

Upregulation of CB2 receptors in reactive astrocytes in canine degenerative myelopathy, a disease model of amyotrophic lateral sclerosis.

Targeting of the CB2 receptor results in neuroprotection in the SOD1G93A mutant mouse model of amyotrophic lateral sclerosis (ALS). The neuroprotective effects of CB2 receptors are facilitated by their upregulation in the spinal cord of the mutant mice. Here, we investigated whether similar CB2 receptor upregulation, as well as parallel changes in other endocannabinoid elements, is evident in the spinal cord of dogs with degenerative myelopathy (DM), caused by mutations in the superoxide dismutase 1 gene (SOD1). We used well-characterized post-mortem spinal cords from unaffected and DM-affected dogs. Tissues were used first to confirm the loss of motor neurons using Nissl staining, which was accompanied by glial reactivity (elevated GFAP and Iba-1 immunoreactivity). Next, we investigated possible differences in the expression of endocannabinoid genes measured by qPCR between DM-affected and control dogs. We found no changes in expression of the CB1 receptor (confirmed with CB1 receptor immunostaining) or NAPE-PLD, DAGL, FAAH and MAGL enzymes. In contrast, CB2 receptor levels were significantly elevated in DM-affected dogs determined by qPCR and western blotting, which was confirmed in the grey matter using CB2 receptor immunostaining. Using double-labelling immunofluorescence, CB2 receptor immunolabelling colocalized with GFAP but not Iba-1, indicating upregulation of CB2 receptors on astrocytes in DM-affected dogs. Our results demonstrate a marked upregulation of CB2 receptors in the spinal cord in canine DM, which is concentrated in activated astrocytes. Such receptors could be used as a potential target to enhance the neuroprotective effects exerted by these glial cells.

The disease-modifying effects of a Sativex-like combination of phytocannabinoids in mice with experimental autoimmune encephalomyelitis are preferentially due to Δ9-tetrahydrocannabinol acting through CB1 receptors.

Sativex(®), an equimolecular combination of Δ(9)-tetrahydrocannabinol-botanical drug substance (Δ(9)-THC-BDS) and cannabidiol-botanical drug substance (CBD-BDS), is a licensed medicine that may be prescribed for alleviating specific symptoms of multiple sclerosis (MS) such as spasticity and pain. However, further evidence suggest that it could be also active as disease-modifying therapy given the immunomodulatory, anti-inflammatory and cytoprotective properties of their two major components. In this study, we investigated this potential in the experimental autoimmune encephalitis (EAE) model of MS in mice. We compared the effect of a Sativex-like combination of Δ(9)-THC-BDS (10 mg/kg) and CBD-BDS (10 mg/kg) with Δ(9)-THC-BDS (20 mg/kg) or CBD-BDS (20 mg/kg) administered separately by intraperitoneal administration to EAE mice. Treatments were initiated at the time that symptoms appear and continued up to the first relapse of the disease. The results show that the treatment with a Sativex-like combination significantly improved the neurological deficits typical of EAE mice, in parallel with a reduction in the number and extent of cell aggregates present in the spinal cord which derived from cell infiltration to the CNS. These effects were completely reproduced by the treatment with Δ(9)-THC-BDS alone, but not by CBD-BDS alone which only delayed the onset of the disease without improving disease progression and reducing the cell infiltrates in the spinal cord. Next, we investigated the potential targets involved in the effects of Δ(9)-THC-BDS by selectively blocking CB(1) or PPAR-γ receptors, and we found a complete reversion of neurological benefits and the reduction in cell aggregates only with rimonabant, a selective CB(1) receptor antagonist. Collectively, our data support the therapeutic potential of Sativex as a phytocannabinoid formulation capable of attenuating EAE progression, and that the active compound was Δ(9)-THC-BDS acting through CB(1) receptors.

Changes in the endocannabinoid signaling system in CNS structures of TDP-43 transgenic mice: relevance for a neuroprotective therapy in TDP-43-related disorders.

Because of their neuroprotective properties, cannabinoids are being investigated in neurodegenerative disorders, mainly in preclinical studies. These disorders also include amyotrophic lateral sclerosis (ALS), a degenerative disease produced by the damage of the upper and lower motor neurons leading to muscle denervation, atrophy and paralysis. The studies with cannabinoids in ALS have been conducted exclusively in a transgenic mouse model bearing mutated forms of human superoxide dismutase-1, the first gene that was identified in relation with ALS. The present study represents the first attempt to investigate the endocannabinoid system in an alternative model, the transgenic mouse model of TAR-DNA binding protein-43 (TDP-43), a protein related to ALS and also to frontotemporal dementia. We used these mice for behavioral and histological characterization at an early symptomatic phase (70-80 days of age) and at a post-symptomatic stage (100-110 days of age). TDP-43 transgenic mice exhibited a worsened rotarod performance at both disease stages. This was accompanied by a loss of motor neurons in the spinal cord (measured by Nissl staining) and by reactive microgliosis (measured by Iba-1 immunostaining) at the post-symptomatic stage. We also detected elevated levels of the CB2 receptor (measured by qRT-PCR and western blotting) in the spinal cord of these animals. Double-staining studies confirmed that this up-regulation occurs in microglial cells in the post-symptomatic stage. Some trends towards an increase were noted also for the levels of endocannabinoids, which in part correlate with a small reduction of FAAH. Some of these parameters were also analyzed in the cerebral cortex of TDP-43 transgenic mice, but we did not observe any significant change, in agreement with the absence of anomalies in cognitive tests. In conclusion, our data support the idea that the endocannabinoid signaling system, in particular the CB2 receptor, may serve for the development of a neuroprotective therapy in TDP-43-related disorders. We are presently engaged in pharmacological experiments to investigate this possibility.

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.

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 Δ(9) -tetrahydrocannabinol- and cannabidiol-enriched botanical extracts (20 mg/kg for each phytocannabinoid). RESULTS: We found a significant increase of CB2 receptors and NAPE-PLD enzyme in SOD1(G93A) transgenic males and only CB2 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 CB2 receptors, in SOD1(G93A) transgenic mice, and provide support that Sativex(®) may serve as a novel disease-modifying therapy in ALS.

Motor effects of the non-psychotropic phytocannabinoid cannabidiol that are mediated by 5-HT1A receptors.

The broad presence of CB1 receptors in the basal ganglia, mainly in GABA- or glutamate-containing neurons, as well as the presence of TRPV1 receptors in dopaminergic neurons and the identification of CB2 receptors in some neuronal subpopulations within the basal ganglia, explain the powerful motor effects exerted by those cannabinoids that can activate/block these receptors. By contrast, cannabidiol (CBD), a phytocannabinoid with a broad therapeutic profile, is generally presented as an example of a cannabinoid compound with no motor effects due to its poor affinity for the CB1 and the CB2 receptor, despite its activity at the TRPV1 receptor. However, recent evidence suggests that CBD may interact with the serotonin 5-HT1A receptor to produce some of its beneficial effects. This may enable CBD to directly influence motor activity through the well-demonstrated role of serotonergic transmission in the basal ganglia. We have investigated this issue in rats using three different pharmacological and neurochemical approaches. First, we compared the motor effects of various i.p. doses of CBD with the selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; i.p.). Second, we investigated whether the motor effects of CBD are sensitive to 5-HT1A receptor blockade in comparison with CB1 receptor antagonism. Finally, we investigated whether CBD was able to potentiate the effect of a sub-effective dose of 8-OH-DPAT. Our results demonstrated that: (i) only high doses of CBD (>10 mg/kg) altered motor behavior measured in a computer-aided actimeter; (ii) these alterations were restricted to vertical activity (rearing) with only modest changes in other parameters; (iii) similar effects were produced by 8-OH-DPAT (1 mg/kg), although this agonist affected exclusively vertical activity, with no effects on other motor parameters, and it showed always more potency than CBD; (iv) the effects of 8-OH-DPAT (1 mg/kg) and CBD (20 mg/kg) on vertical activity were reversed by the 5-HT1A receptor antagonist WAY-100,635 (0.5 mg/kg; i.p.); (v) the effects of CBD (20 mg/kg) on vertical activity were not reversed by the CB1 receptor antagonist rimonabant (0.1 mg/kg; i.p.); (vi) the effect of 8-OH-DPAT on vertical activity was associated with an increase in serotonin content in the basal ganglia, a neurochemical change not produced by CBD (20 mg/kg); and (vii) CBD at a dose of 20 mg/kg was able to enhance motor effects of a sub-effective dose of 8-OH-DPAT (0.1 mg/kg), producing the expected changes in serotonergic transmission in the basal ganglia. Collectively, these results suggest that CBD may influence motor activity, in particular vertical activity, and that this effect seems to be dependent on its ability to target the 5-HT1A receptor, a mechanism of action that has been proposed to account for its anti-emetic, anxiolytic and antidepressant effects.