Preclinical investigation in FAAH inhibition as a neuroprotective therapy for frontotemporal dementia using TDP-43 transgenic male mice.

BACKGROUND: Frontotemporal dementia (FTD) is a heterogeneous group of early onset and progressive neurodegenerative disorders, characterized by degeneration in the frontal and temporal lobes, which causes deterioration in cognition, personality, social behavior and language. Around 45% of the cases are characterized by the presence of aggregates of the RNA-binding protein TDP-43. METHODS: In this study, we have used a murine model of FTD that overexpresses this protein exclusively in the forebrain (under the control of the CaMKIIα promoter) for several biochemical, histological and pharmacological studies focused on the endocannabinoid system. RESULTS: These mice exhibited at postnatal day 90 (PND90) important cognitive deficits, signs of emotional impairment and disinhibited social behaviour, which were, in most of cases, maintained during the first year of life of these animals. Motor activity was apparently normal, but FTD mice exhibited higher mortality. Their MRI imaging analysis and their ex-vivo histopathological evaluation proved changes compatible with atrophy (loss of specific groups of pyramidal neurons: Ctip2- and NeuN-positive cells) and inflammatory events (astroglial and microglial reactivities) in both cortical (medial prefrontal cortex) and subcortical (hippocampus) structures at PND90 and also at PND365. The analysis of the endocannabinoid system in these mice proved a decrease in the hydrolysing enzyme FAAH in the prefrontal cortex and the hippocampus, with an increase in the synthesizing enzyme NAPE-PLD only in the hippocampus, responses that were accompanied by modest elevations in anandamide and related N-acylethanolamines. The potentiation of these elevated levels of anandamide after the pharmacological inactivation of FAAH with URB597 resulted in a general improvement in behaviour, in particular in cognitive deterioration, associated with the preservation of pyramidal neurons of the medial prefrontal cortex and the CA1 layer of the hippocampus, and with the reduction of gliosis in both structures. CONCLUSIONS: Our data confirmed the potential of elevating the endocannabinoid tone as a therapy against TDP-43-induced neuropathology in FTD, limiting glial reactivity, preserving neuronal integrity and improving cognitive, emotional and social deficits.

BiP Heterozigosity Aggravates Pathological Deterioration in Experimental Amyotrophic Lateral Sclerosis.

In the present study, we investigated the involvement of the chaperone protein BiP (also known as GRP78 or Hspa5), a master regulator of intracellular proteostasis, in two mouse models of neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). To this end, we used mice bearing partial genetic deletion of the BiP gene (BiP+/- mice), which, for the ALS model, were crossed with mutant SOD1 (mSOD1) transgenic mice to generate mSOD1/BiP+/- double mutant mice. Our data revealed a more intense neurological decline in the double mutants, reflected in a greater deterioration of the neurological score and rotarod performance, with also a reduced animal survival, compared to mSOD1 transgenic mice. Such worsening was associated with higher microglial (labelled with Iba-1 immunostaining) and, to a lesser extent, astroglial (labelled with GFAP immunostaining) immunoreactivities found in the double mutants, but not with a higher loss of spinal motor neurons (labelled with Nissl staining) in the spinal cord. The morphological analysis of Iba-1 and GFAP-positive cells revealed a higher presence of activated cells, characterized by elevated cell body size and shorter processes, in double mutants compared to mSOD1 mice with normal BiP expression. In the case of the PD model, BiP+/- mice were unilaterally lesioned with the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). In this case, however, we did not detect a greater susceptibility to damage in mutant mice, as the motor defects caused by 6-OHDA in the pole test and the cylinder rearing test, as well as the losses in tyrosine hydroxylase-containing neurons and the elevated glial reactivity (labelled with CD68 and GFAP immunostaining) detected in the substantia nigra were of similar magnitude in BiP+/- mice compared with wildtype animals. Therefore, our findings support the view that a dysregulation of the protein BiP may contribute to ALS pathogenesis. As BiP has been recently related to cannabinoid type-1 (CB1) receptor function, our work also opens the door to future studies on a possible link between BiP and the neuroprotective effects of cannabinoids that have been widely reported in this neuropathological context. In support of this possibility, preliminary data indicate that CB1 receptor levels are significantly reduced in mSOD1 mice having partial deletion of BiP gene.

Preclinical Investigation in Neuroprotective Effects of the GPR55 Ligand VCE-006.1 in Experimental Models of Parkinson's Disease and Amyotrophic Lateral Sclerosis.

Cannabinoids act as pleiotropic compounds exerting, among others, a broad-spectrum of neuroprotective effects. These effects have been investigated in the last years in different preclinical models of neurodegeneration, with the cannabinoid type-1 (CB1) and type-2 (CB2) receptors concentrating an important part of this research. However, the issue has also been extended to additional targets that are also active for cannabinoids, such as the orphan G-protein receptor 55 (GPR55). In the present study, we investigated the neuroprotective potential of VCE-006.1, a chromenopyrazole derivative with biased orthosteric and positive allosteric modulator activity at GPR55, in murine models of two neurodegenerative diseases. First, we proved that VCE-006.1 alone could induce ERK1/2 activation and calcium mobilization, as well as increase cAMP response but only in the presence of lysophosphatidyl inositol. Next, we investigated this compound administered chronically in two neurotoxin-based models of Parkinson's disease (PD), as well as in some cell-based models. VCE-006.1 was active in reversing the motor defects caused by 6-hydroxydopamine (6-OHDA) in the pole and the cylinder rearing tests, as well as the losses in tyrosine hydroxylase-containing neurons and the elevated glial reactivity detected in the substantia nigra. Similar cytoprotective effects were found in vitro in SH-SY5Y cells exposed to 6-OHDA. We also investigated VCE-006.1 in LPS-lesioned mice with similar beneficial effects, except against glial reactivity and associated inflammatory events, which remained unaltered, a fact confirmed in BV2 cells treated with LPS and VCE-006.1. We also analyzed GPR55 in these in vivo models with no changes in its gene expression, although GPR55 was down-regulated in BV2 cells treated with LPS, which may explain the lack of efficacy of VCE-006.1 in such an assay. Furthermore, we investigated VCE-006.1 in two genetic models of amyotrophic lateral sclerosis (ALS), mutant SOD1, or TDP-43 transgenic mice. Neither the neurological decline nor the deteriorated rotarod performance were prevented with this compound, and the same happened with the elevated microglial and astroglial reactivities, albeit modest spinal motor neuron preservation was achieved in both models. We also analyzed GPR55 in these in vivo models and found no changes in both TDP-43 transgenic and mSOD1 mice. Therefore, our findings support the view that targeting the GPR55 may afford neuroprotection in experimental PD, but not in ALS, thus stressing the specificities for the development of cannabinoid-based therapies in the different neurodegenerative disorders.

Endocannabinoid-hydrolysing enzymes in the post-mortem cerebellum of humans affected by hereditary autosomal dominant ataxias.

OBJECTIVES: Spinocerebellar ataxias (SCAs) are characterized by a loss of balance and motor coordination due to degeneration of the cerebellum and its afferent and efferent connections. We recently found important changes in cannabinoid CB1 and CB2 receptors in the post-mortem cerebellum of patients affected by different SCAs. METHODS: We wanted to further explore this issue by analysing the two major endocannabinoid-hydrolysing enzymes, fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL), in the post-mortem cerebellum of SCA patients and control subjects. RESULTS: Immunoreactivity for the FAAH and MAGL enzymes was found in the granular layer, in Purkinje cells, in neurons of the dentate nucleus and in areas of white matter in the cerebellum of patients at levels frequently notably higher than those in control subjects. Using double-labelling procedures, we found co-localization of FAAH and MAGL with calbindin, supporting the presence of these enzymes in Purkinje neurons. CONCLUSIONS: Degradative endocannabinoid enzymes are significantly increased in the cerebellum of SCA patients, which would presumably lead to reduced endocannabinoid levels. The identification of these enzymes in Purkinje neurons suggests a relationship with the pathogenesis of SCAs and suggests that the endocannabinoid system could provide potential therapeutic targets for the treatment of disease progression in SCAs.

Involvement of CB1 and CB2 receptors in neuroprotective effects of cannabinoids in experimental TDP-43 related frontotemporal dementia using male mice.

BACKGROUND: The elevation of endocannabinoid levels through inhibiting their degradation afforded neuroprotection in CaMKIIα-TDP-43 mice, a conditional transgenic model of frontotemporal dementia. However, which cannabinoid receptors are mediating these benefits is still pending to be elucidated. METHODS: We have investigated the involvement of the CB1 and the CB2 receptor using chronic treatments with selective ligands in CaMKIIα-TDP-43 mice, analysis of their cognitive deterioration with the Novel Object Recognition test, and immunostaining for neuronal and glial markers in two areas of interest in frontotemporal dementia. RESULTS: Our results confirmed the therapeutic value of activating either the CB1 or the CB2 receptor, with improvements in the animal performance in the Novel Object Recognition test, preservation of pyramidal neurons, in particular in the medial prefrontal cortex, and attenuation of glial reactivity, in particular in the hippocampus. In addition, the activation of both CB1 and CB2 receptors reduced the elevated levels of TDP-43 in the medial prefrontal cortex of CaMKIIα-TDP-43 mice, an effect exerted by mechanisms that are currently under investigation. CONCLUSIONS: These data reinforce the notion that the activation of CB1 and CB2 receptors may represent a promising therapy against TDP-43-induced neuropathology in frontotemporal dementia. Future studies will have to confirm these benefits, in particular with one of the selective CB2 agonists used here, which has been thoroughly characterized for clinical development.

Endocannabinoid-Binding Receptors as Drug Targets.

Cannabis plant has been used from ancient times with therapeutic purposes for treating human pathologies, but the identification of the cellular and molecular mechanisms underlying the therapeutic properties of the phytocannabinoids, the active compounds in this plant, occurred in the last years of the past century. In the late 1980s and early 1990s, seminal studies demonstrated the existence of cannabinoid receptors and other elements of the so-called endocannabinoid system. These G protein-coupled receptors (GPCRs) are a key element in the functions assigned to endocannabinoids and appear to serve as promising pharmacological targets. They include CB1, CB2, and GPR55, but also non-GPCRs can be activated by endocannabinoids, like ionotropic receptor TRPV1 and even nuclear receptors of the PPAR family. Their activation, inhibition, or simply modulation have been associated with numerous physiological effects at both central and peripheral levels, which may have therapeutic value in different human pathologies, then providing a solid experimental explanation for both the ancient medicinal uses of Cannabis plant and the recent advances in the development of cannabinoid-based specific therapies. This chapter will review the scientific knowledge generated in the last years around the research on the different endocannabinoid-binding receptors and their signaling mechanisms. Our intention is that this knowledge may help readers to understand the relevance of these receptors in health and disease conditions, as well as it may serve as the theoretical basis for the different experimental protocols to investigate these receptors and their signaling mechanisms that will be described in the following chapters.

Inactivation of the CB2 receptor accelerated the neuropathological deterioration in TDP-43 transgenic mice, a model of amyotrophic lateral sclerosis.

The activation of the cannabinoid receptor type-2 (CB2 ) afforded neuroprotection in amyotrophic lateral sclerosis (ALS) models. The objective of this study was to further investigate the relevance of the CB2 receptor through investigating the consequences of its inactivation. TDP-43(A315T) transgenic mice were crossed with CB2 receptor knock-out mice to generate double mutants. Temporal and qualitative aspects of the pathological phenotype of the double mutants were compared to TDP-43 transgenic mice expressing the CB2 receptor. The double mutants exhibited significantly accelerated neurological decline, such that deteriorated rotarod performance was visible at 7 weeks, whereas rotarod performance was normal up to 11 weeks in transgenic mice with intact expression of the CB2 receptor. A morphological analysis of spinal cords confirmed an earlier death (visible at 65 days) of motor neurons labelled with Nissl staining and ChAT immunofluorescence in double mutants compared to TDP-43 transgenic mice expressing the CB2 receptor. Evidence of glial reactivity, measured using GFAP and Iba-1 immunostaining, was seen in double mutants at 65 days, but not in TDP-43 transgenic mice expressing the CB2 receptor. However, at 90 days, both genotypes exhibited similar changes for all these markers, although surviving motor neurons of transgenic mice presented some morphological abnormalities in absence of the CB2 receptor that were not as evident in the presence of this receptor. This faster deterioration seen in double mutants led to premature mortality compared with TDP-43 transgenic mice expressing the CB2 receptor. We also investigated the consequences of a pharmacological inactivation of the CB2 receptor using the selective antagonist AM630 in TDP-43 transgenic mice, but results showed only subtle trends towards a greater deterioration. In summary, our results confirmed the potential of the CB2 receptor agonists as a neuroprotective therapy in ALS and strongly support the need to progress towards an evaluation of this potential in patients.

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.

Motor neuron preservation and decrease of in vivo TDP-43 phosphorylation by protein CK-1δ kinase inhibitor treatment.

Pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease where no treatment exists, involves the compartmentalization of the nuclear protein TDP-43 (TAR DNA-binding protein 43) in the cytoplasm which is promoted by its aberrant phosphorylation and others posttranslational modifications. Recently, it was reported that CK-1δ (protein casein kinase-1δ) is able to phosphorylate TDP-43. Here, the preclinical efficacy of a benzothiazole-based CK-1δ inhibitor IGS-2.7, both in a TDP-43 (A315T) transgenic mouse and in a human cell-based model of ALS, is shown. Treatment with IGS-2.7 produces a significant preservation of motor neurons in the anterior horn at lumbar level, a decrease in both astroglial and microglial reactivity in this area, and in TDP-43 phosphorylation in spinal cord samples. Furthermore, the recovery of TDP-43 homeostasis (phosphorylation and localization) in a human-based cell model from ALS patients after treatment with IGS-2.7 is also reported. Moreover, we have shown a trend to increase in CK-1δ mRNA in spinal cord and significantly in frontal cortex of sALS cases. All these data show for the first time the in vivo modulation of TDP-43 toxicity by CK-1δ inhibition with IGS-2.7, which may explain the benefits in the preservation of spinal motor neurons and point to the relevance of CK-1δ inhibitors in a future disease-modifying treatment for ALS.

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.

Endocannabinoid System in Spinocerebellar Ataxia Type-3 and Other Autosomal-Dominant Cerebellar Ataxias: Potential Role in Pathogenesis and Expected Relevance as Neuroprotective Targets.

Spinocerebellar ataxias (SCAs) are a group of hereditary and progressive neurological disorders characterized by a loss of balance and motor coordination typically associated with cerebellar atrophy. The most prevalent SCA types are all polyQ disorders like Huntington's disease, sharing the most relevant events in pathogenesis with this basal ganglia disorder, but with most of the damage concentrated in cerebellar neurons, and in their afferent and efferent connections (e.g., brainstem nuclei). SCAs have no cure and effective symptom-alleviating and disease-modifying therapies are not currently available. However, based on results obtained in studies conducted in murine models and information derived from analyses in post-mortem tissue samples from patients, which show notably higher levels of CB1 receptors found in different cerebellar neuronal subpopulations, the blockade of these receptors has been proposed for acutely modulating motor incoordination in cerebellar ataxias, whereas their chronic activation has been proposed for preserving specific neuronal losses. Additional studies in post-mortem tissues from SCA patients have also demonstrated elevated levels of CB2 receptors in Purkinje neurons as well as in glial elements in the granular layer and in the cerebellar white matter, with a similar profile found for endocannabinoid hydrolyzing enzymes, then suggesting that activating CB2 receptors and/or inhibiting these enzymes may also serve to develop cannabinoid-based neuroprotective therapies. The present review will address both aspects. On one hand, the endocannabinoid system becomes dysregulated in the cerebellum and also in other CNS structures (e.g., brainstem, basal ganglia) in SCAs, which may contribute to the progression of pathogenic events in these diseases. On the other hand, these endocannabinoid alterations may be pharmacologically corrected or enhanced, and this may have therapeutic consequences, either alleviating specific symptoms or eliciting neuroprotective effects, an objective presently under investigation.

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.

Altered striatal endocannabinoid signaling in a transgenic mouse model of spinocerebellar ataxia type-3.

Spinocerebellar ataxia type-3 (SCA-3) is the most prevalent autosomal dominant inherited ataxia. We recently found that the endocannabinoid system is altered in the post-mortem cerebellum of SCA-3 patients, and similar results were also found in the cerebellar and brainstem nuclei of a SCA-3 transgenic mouse model. Given that the neuropathology of SCA-3 is not restricted to these two brain regions but rather, it is also evident in other structures (e.g., the basal ganglia), we studied the possible changes to endocannabinoid signaling in the striatum of these transgenic mice. SCA-3 mutant mice suffer defects in motor coordination, balance and they have an abnormal gait, reflecting a cerebellar/brainstem neuropathology. However, they also show dystonia-like behavior (limb clasping) that may be related to the malfunction/deterioration of specific neurons in the striatum. Indeed, we found a loss of striatal projecting neurons in SCA-3 mutant mice, accompanied by a reduction in glial glutamate transporters that could potentially aggravate excitotoxic damage. In terms of endocannabinoid signaling, no changes in CB2 receptors were evident, yet an important reduction in CB1 receptors was detected by qPCR and immunostaining. The reduction in CB1 receptors was presumed to occur in striatal afferent and efferent neurons, also potentially aggravating excitotoxicity. We also measured the endocannabinoid lipids in the striatum and despite a marked increase in the FAAH enzyme in this area, no overall changes in these lipids were found. Collectively, these studies confirm that the striatal endocannabinoid system is altered in SCA-3 mutant mice, adding to the equivalent changes found in other strongly affected CNS structures in this type of ataxia (i.e.: the cerebellum and brainstem). These data open the way to search for drugs that might correct these changes.

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.

New pyridazinone-4-carboxamides as new cannabinoid receptor type-2 inverse agonists: Synthesis, pharmacological data and molecular docking.

In the last few years, cannabinoid type-2 receptor (CB2R) selective ligands have shown a great potential as novel therapeutic drugs in several diseases. With the aim of discovering new selective cannabinoid ligands, a series of pyridazinone-4-carboxamides was designed and synthesized, and the new derivatives tested for their affinity toward the hCB1R and hCB2R. The 6-(4-chloro-3-methylphenyl)-2-(4-fluorobenzyl)-N-(cis-4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide (9) displayed high CB2-affinity (KiCB2 = 2.0 ± 0.81 nM) and a notable selectivity (KiCB1/KiCB2 > 2000). In addition, 9 and other active new synthesized entities have demonstrated to behave as CB2R inverse agonists in [35S]-GTPγS binding assay. ADME predictions of the newly synthesized CB2R ligands suggest a favourable pharmacokinetic profile. Docking studies disclosed the specific pattern of interactions of these derivatives. Our results support that pyridazinone-4-carboxamides represent a new promising scaffold for the development of potent and selective CB2R ligands.

Targeting Cannabinoid CB2 Receptors in the Central Nervous System. Medicinal Chemistry Approaches with Focus on Neurodegenerative Disorders.

Endocannabinoids activate two types of specific G-protein-coupled receptors (GPCRs), namely cannabinoid CB1 and CB2. Contrary to the psychotropic actions of agonists of CB1 receptors, and serious side effects of the selective antagonists of this receptor, drugs acting on CB2 receptors appear as promising drugs to combat CNS diseases (Parkinson's disease, Huntington's chorea, cerebellar ataxia, amyotrohic lateral sclerosis). Differential localization of CB2 receptors in neural cell types and upregulation in neuroinflammation are keys to understand the therapeutic potential in inter alia diseases that imply progressive neurodegeneration. Medicinal chemistry approaches are now engaged to develop imaging tools to map receptors in the living human brain, to develop more efficacious agonists, and to investigate the possibility to develop allosteric modulators.

Dysregulation of the endocannabinoid signaling system in the cerebellum and brainstem in a transgenic mouse model of spinocerebellar ataxia type-3.

Spinocerebellar ataxia type-3 (SCA-3) is a rare disease but it is the most frequent type within the autosomal dominant inherited ataxias. The disease lacks an effective treatment to alleviate major symptoms and to modify disease progression. Our recent findings that endocannabinoid receptors and enzymes are significantly altered in the post-mortem cerebellum of patients affected by autosomal-dominant hereditary ataxias suggest that targeting the endocannabinoid signaling system may be a promising therapeutic option. Our goal was to investigate the status of the endocannabinoid signaling system in a transgenic mouse model of SCA-3, in the two CNS structures most affected in this disease - cerebellum and brainstem. These animals exhibited progressive motor incoordination, imbalance, abnormal gait, muscle weakness, and dystonia, in parallel to reduced in vivo brain glucose metabolism, deterioration of specific neuron subsets located in the dentate nucleus and pontine nuclei, small changes in microglial morphology, and reduction in glial glutamate transporters. Concerning the endocannabinoid signaling, our data indicated no changes in CB2 receptors. By contrast, CB1 receptors increased in the Purkinje cell layer, in particular in terminals of basket cells, but they were reduced in the dentate nucleus. We also measured the levels of endocannabinoid lipids and found reductions in anandamide and oleoylethanolamide in the brainstem. These changes correlated with an increase in the FAAH enzyme in the brainstem, which also occurred in some cerebellar areas, whereas other endocannabinoid-related enzymes were not altered. Collectively, our results in SCA-3 mutant mice confirm a possible dysregulation in the endocannabinoid system in the most important brain structures affected in this type of ataxia, suggesting that a pharmacological manipulation addressed to correct these changes could be a promising option in SCA-3.

Changes in CB(1) and CB(2) receptors in the post-mortem cerebellum of humans affected by spinocerebellar ataxias.

BACKGROUND AND PURPOSE: Spinocerebellar ataxias (SCAs) are a family of chronic progressive neurodegenerative diseases, clinically and genetically heterogeneous, characterized by loss of balance and motor coordination due to degeneration of the cerebellum and its afferent and efferent connections. Unlike other motor disorders, the possible role of changes in the endocannabinoid system in the pathogenesis of SCAs has not been investigated. EXPERIMENTAL APPROACH: The status of cannabinoid receptor type 1 (CB1 ) and cannabinoid receptor type 2 (CB2 ) receptors in the post-mortem cerebellum of SCA patients and controls was investigated using immunohistochemical procedures. KEY RESULTS: Immunoreactivity for the CB1 receptor, and also for the CB2 receptor, was found in the granular layer, Purkinje cells, neurons of the dentate nucleus and areas of white matter in the cerebellum of SCA patients at levels notably higher than controls. Double-labelling procedures demonstrated co-localization of CB1 and, in particular, CB2 receptors with calbindin, supporting the presence of these receptors in Purkinje neurons. Both receptors also co-localized with Iba-1 and glial fibrillary acidic protein in the granular layer and white matter areas, indicating that they are present in microglia and astrocytes respectively. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that CB1 and CB2 receptor levels are significantly altered in the cerebellum of SCA patients. Their identification in Purkinje neurons, which are the main cells affected in SCAs, as well as the changes they experienced, suggest that alterations in endocannabinoid receptors may be related to the pathogenesis of SCAs. Therefore, the endocannabinoid system could provide potential therapeutic targets for the treatment of SCAs and its progression. LINKED ARTICLES: This article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6.