Isabel Lastres-Becker

Cannabinoids provide neuroprotection against 6-hydroxydopamine toxicity in vivo and in vitro: Relevance to Parkinson's disease

Cannabinoids have been reported to provide neuroprotection in acute and chronic neurodegeneration. In this study, we examined whether they are also effective against the toxicity caused by 6-hydroxydopamine, both in vivo and in vitro, which may be relevant to Parkinsons disease (PD). First, we evaluated whether the administration of cannabinoids in vivo reduces the neurodegeneration produced by a unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. As expected, 2 weeks after the application of this toxin, a significant depletion of dopamine contents and a reduction of tyrosine hydroxylase activity in the lesioned striatum were noted, and were accompanied by a reduction in tyrosine hydroxylase-mRNA levels in the substantia nigra. None of these events occurred in the contralateral structures. Daily administration of delta9-tetrahydrocannabinol (delta9-THC) during these 2 weeks produced a significant waning in the magnitude of these reductions, whereas it failed to affect dopaminergic parameters in the contralateral structures. This effect of delta9-THC appeared to be irreversible since interruption of the daily administration of this cannabinoid after the 2-week period did not lead to the re-initiation of the 6-hydroxydopamine-induced neurodegeneration. In addition, the fact that the same neuroprotective effect was also produced by cannabidiol (CBD), another plant-derived cannabinoid with negligible affinity for cannabinoid CB1 receptors, suggests that the antioxidant properties of both compounds, which are cannabinoid receptor-independent, might be involved in these in vivo effects, although an alternative might be that the neuroprotection exerted by both compounds might be due to their anti-inflammatory potential. As a second objective, we examined whether cannabinoids also provide neuroprotection against the in vitro toxicity of 6-hydroxydopamine. We found that the non-selective cannabinoid agonist HU-210 increased cell survival in cultures of mouse cerebellar granule cells exposed to this toxin. However, this effect was significantly lesser when the cannabinoid was directly added to neuronal cultures than when these cultures were exposed to conditioned medium obtained from mixed glial cell cultures treated with HU-210, suggesting that the cannabinoid exerted its major protective effect by regulating glial influence to neurons. In summary, our results support the view of a potential neuroprotective action of cannabinoids against the in vivo and in vitro toxicity of 6-hydroxydopamine, which might be relevant for PD. Our data indicated that these neuroprotective effects might be due, among others, to the antioxidant properties of certain plant-derived cannabinoids, or exerted through the capability of cannabinoid agonists to modulate glial function, or produced by a combination of both mechanisms.

Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration

Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N‐arachidonoylethanolamine (anandamide) and 2‐arachidonoylglycerol (2‐AG), as well as other constituents of their lipid families, N‐acylethanolamines (NAEs) and 2‐monoacylglycerols (2‐MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46‐fold increase of cortical NAE concentrations (anandamide, 13‐fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2‐AG and other 2‐MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB1 receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up‐regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2‐AG, and CB1 receptor density.

The endogenous cannabinoid system and the basal ganglia: biochemical, pharmacological, and therapeutic aspects

New data strengthen the idea of a prominent role for endocannabinoids in the modulation of a wide variety of neurobiological functions. Among these, one of the most important is the control of movement. This finding is supported by 3 lines of evidence: (1) the demonstration of a powerful action, mostly inhibitory in nature, of synthetic and plant-derived cannabinoids and, more recently, of endocannabinoids on motor activity; (2) the presence of the cannabinoid CB(1) receptor subtype and the recent description of endocannabinoids in the basal ganglia and the cerebellum, the areas that control movement; and (3) the fact that CB(1) receptor binding was altered in the basal ganglia of humans affected by several neurological diseases and also of rodents with experimentally induced motor disorders. Based on this evidence, it has been suggested that new synthetic compounds that act at key steps of endocannabinoid activity (i.e., more-stable analogs of endocannabinoids, inhibitors of endocannabinoid reuptake or metabolism, antagonists of CB(1) receptors) might be of interest for their potential use as therapeutic agents in a variety of pathologies affecting extrapyramidal structures, such as Parkinsons and Huntingtons diseases. Currently, only a few data exist in the literature studying such relationships in humans, but an increasing number of journal articles are revealing the importance of this new neuromodulatory system and arguing in favour of the funding of more extensive research in this field. The present article will review the current knowledge of this neuromodulatory system, trying to establish the future lines for research on the therapeutic potential of the endocannabinoid system in motor disorders.

Hypolocomotor effects in rats of capsaicin and two long chain capsaicin homologues.

Capsaicin and its analogue N-arachidonoyl-vanillyl-amine (arvanil) are agonists of vanilloid VR1 receptors, and suppress spontaneous activity in mice through an unknown mechanism. Here, we tested in rats the effect on motor behavior of: (1) capsaicin; (2) N-linoleoyl-vanillyl-amine (livanil) and N-alpha-linolenoyl-vanillyl-amine (linvanil), which, unlike arvanil, have very little affinity for cannabinoid CB1 receptors; and (3) the endocannabinoid anandamide (N-arachidonoyl-ethanolamine), which is a full agonist at both cannabinoid CB1 and vanilloid VR1 receptors. All compounds, administered i.p., dose-dependently (0.1-10 mg/kg) inhibited ambulation and stereotypic behavior and increased inactivity in the open field test. The rank of potency observed in vivo (livanil>capsaicin>linvanil>anandamide) bore little resemblance with the relative potencies in a functional assay for rat vanilloid VR1 receptors (livanil=linvanil>capsaicin>anandamide) and even less with the relative affinities in rat CB1 receptor binding assays (anandamide>livanil>linvanil>capsaicin). The vanilloid VR1 receptor antagonist capsazepine (10 mg/kg, i.p.) blocked the effect of capsaicin but not of livanil or anandamide, whereas the CB1 receptor antagonist (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.HCl (SR141716A, 3 mg/kg, i.p.) antagonized the actions of the CB1 receptor agonist Delta9-tetrahydrocannabinol, but not of livanil, anandamide or capsaicin. Anandamide occluded the effects of livanil on locomotion, possibly suggestive of a common mechanism of action for the two compounds. Finally, stimulation with capsaicin of cells expressing rat vanilloid VR1 receptors led to anandamide formation. These data suggest that motor behavior can be suppressed by the activation of: (1) vanilloid receptors, possibly via the intermediacy of anandamide; or (2) capsazepine- and SR141716A-insensitive sites of action for anandamide, livanil and linvanil, possibly the same that were previously suggested to mediate arvanil hypokinetic effects in mice.

Cannabinoid CB2 receptor agonists protect the striatum against malonate toxicity: relevance for Huntington's disease.

Cannabinoid agonists might serve as neuroprotective agents in neurodegenerative disorders. Here, we examined this hypothesis in a rat model of Huntingtons disease (HD) generated by intrastriatal injection of the mitochondrial complex II inhibitor malonate. Our results showed that only compounds able to activate CB2 receptors were capable of protecting striatal projection neurons from malonate‐induced death. That CB2 receptor agonists are neuroprotective was confirmed by using the selective CB2 receptor antagonist, SR144528, and by the observation that mice deficient in CB2 receptor were more sensitive to malonate than wild‐type animals. CB2 receptors are scarce in the striatum in healthy conditions, but they are markedly upregulated after the lesion with malonate. Studies of double immunostaining revealed a significant presence of CB2 receptors in cells labeled with the marker of reactive microglia OX‐42, and also in cells labeled with GFAP (a marker of astrocytes). We further showed that the activation of CB2 receptors significantly reduced the levels of tumor necrosis factor‐α (TNF‐α) that had been increased by the lesion with malonate. In summary, our results demonstrate that stimulation of CB2 receptors protect the striatum against malonate toxicity, likely through a mechanism involving glial cells, in particular reactive microglial cells in which CB2 receptors would be upregulated in response to the lesion. Activation of these receptors would reduce the generation of proinflammatory molecules like TNF‐α. Altogether, our results support the hypothesis that CB2 receptors could constitute a therapeutic target to slowdown neurodegeneration in HD.

Compounds acting at the endocannabinoid and/or endovanilloid systems reduce hyperkinesia in a rat model of Huntington's disease

We have recently reported that the administration of AM404, an inhibitor of the endocannabinoid re‐uptake process, which also has affinity for the vanilloid VR1 receptors, is able to reduce hyperkinesia, and causes recovery from neurochemical deficits, in a rat model of Huntingtons disease (HD) generated by bilateral intrastriatal injections of 3‐nitropropionic acid (3NP). In the present study, we wanted to explore the mechanism(s) by which AM404 produces its antihyperkinetic effect in 3NP‐lesioned rats by employing several experimental approaches. First, we tried to block the effects of AM404 with selective antagonists for the CB1 or VR1 receptors, i.e. SR141716A and capsazepine, respectively. We found that the reduction caused by AM404 of the increased ambulation exhibited by 3NP‐lesioned rats in the open‐field test was reversed when the animals had been pre‐treated with capsazepine but not with SR141716A, thus suggesting a major role of VR1 receptors in the antihyperkinetic effects of AM404. However, despite the lack of behavioral effects of the CB1 receptor antagonist, the pretreatment with this compound abolished the recovery of neurochemical [γ‐aminobutyric acid (GABA) and dopamine] deficits in the caudate‐ putamen caused by AM404, as also did capsazepine. In a second group of studies, we wanted to explore the potential antihyperkinetic effects of various compounds which, compared to AM404, exhibit more selectivity for either the endovanilloid or the endocannabinoid systems. First, we tested VDM11 or AM374, two selective inhibitors or the endocannabinoid re‐uptake or hydrolysis, respectively. Both compounds were mostly unable to reduce hyperkinesia in 3NP‐lesioned rats, although VDM11 produced a certain motor depression, and AM374 exhibited a trend to stimulate ambulation, in control rats. We also tested the effects of selective direct agonists for VR1 (capsaicin) or CB1 (CP55,940) receptors. Capsaicin exhibited a strong antihyperkinetic activity and, moreover, was able to attenuate the reductions in dopamine and GABA transmission provoked by the 3NP lesion, whereas CP55,940 had also antihyperkinetic activity but was unable to cause recovery of either dopamine or GABA deficits in the basal ganglia. In summary, our data indicate a major role for VR1 receptors, as compared to CB1 receptors, in the antihyperkinetic effects and the recovery of neurochemical deficits caused in 3NP‐lesioned rats by compounds that activate both CB1 and VR1 receptors, either directly or via manipulation of the levels of endogenous agonists.

Loss of mRNA levels, binding and activation of GTP-binding proteins for cannabinoid CB1 receptors in the basal ganglia of a transgenic model of Huntington's disease.

Data obtained from the basal ganglia of postmortem Huntingtons disease (HD) brains have revealed that the level of cannabinoid CB1 receptors in striatal efferent neurons decreases in parallel to the dysfunction and subsequent degeneration of these neurons. These findings, and others from rat models of HD generated by lesions with mitochondrial toxins, suggest that the loss of CB1 receptors may be involved in the pathogenesis of the disease. To explore further the changes in the endocannabinoid system, as well as the potential of endocannabinoid-related compounds, we examined the status of CB1 receptors in the HD94 transgenic mouse model of HD. These mice express huntingtin exon 1 with a polyglutamine tract of 94 repeats in a tissue-specific and conditional manner using the tet regulatable system. They develop many features of HD, such as striatal atrophy, intraneuronal aggregates and progressive dystonia. In these animals, we analyzed mRNA levels for the CB1 receptor, in addition to the number of specific binding sites and the activation of GTP-binding proteins by CB1 receptor agonists. mRNA transcripts of the CB1 receptor were significantly decreased in the caudate-putamen of HD transgenic mice compared to age-matched littermate controls. The decrease concurred with a marked reduction in receptor density in both the caudate-putamen and its projection areas such as the globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata. Furthermore, the efficacy of CB1 receptor activation was reduced in the globus pallidus, as determined by agonist-induced [35S]GTPgammaS binding, and tended towards a decrease in the substantia nigra. None of these changes was seen in the cerebral cortex and hippocampus, despite high levels of expression of the mutant protein in these regions. The decrease in CB1 receptor levels was accompanied by a decrease in the proenkephalin-mRNA levels but not in substance P-mRNA levels. Taken together, these results suggest that the loss of CB1 receptor might be preferential to the enkephalinergic CB1 receptor-containing striatopallidal neurons, and further implicate the CB1 receptor to the subsequent HD symptomatology and neuropathology.

Extrapyramidal and neuroendocrine effects of AM404, an inhibitor of the carrier-mediated transport of anandamide.

A selective inhibitor of the carrier-mediated transport of endogenous cannabinoids, N-(4-hydroxyphenyl)-arachidonylethanolamide (AM404), has been recently synthesized and proposed as a useful tool for studying the physiological effects of endogenous cannabinoids and as a potential therapeutic agent in a variety of diseases. In the present study, we have examined the effects of this compound in two important brain processes in which a role for anandamide and other endogenous cannabinoids has been claimed: neuroendocrine regulation and extrapyramidal motor activity. A single and well-characterized dose of AM404, which presumably resulted in a significant elevation of the levels of endogenous cannabinoids, produced a marked decrease in plasma prolactin (PRL) levels, with no changes in luteinizing hormone (LH) levels. This decrease in PRL levels was accompanied by an increase in the activity of tyrosine hydroxylase (TH) in the medial basal hypothalamus. Both decreased PRL secretion and increased hypothalamic TH activity have been reported to occur after the administration of anandamide. Administration of AM404 also produced a marked motor inhibition in the open-field test, as also reported for anandamide, with a decrease in ambulatory and exploratory activities and an increase in the time spent in inactivity. This was accompanied by a decrease in the activity of TH in the substantia nigra, an effect also previously observed for anandamide.

The Endocannabinoid System and Huntingtons Disease

The research in Huntingtons disease (HD) has been growing exponentially during the last decade, since the discovery of the genetic basis that leads to neurodegeneration. HD is one of several progressive neurodegenerative disorders, in which the underlying mutation is a CAG expansion encoding a polyglutamine tract in a specific protein, which in the case of HD, is called huntingtin. The first clinical symptoms of HD are generally psychiatric abnormalities, most commonly depression and mood disturbances. Involuntary choreiform movements and dementia develop over the next 15-20 years, and death generally results from complications derived from immobility. There is currently no cure, or even an effective therapy to offset the decline in mental and motor capabilities suffered by those affected by HD, but recent studies have started to examine the usefulness of different classes of new compounds. Among these, plant-derived, synthetic or endogenous cannabinoids have been proposed to have therapeutic value for the treatment of HD, since they act on cannabinoid CB(1) receptors located in the basal ganglia circuitry, that is affected by the striatal atrophy typical of HD. Recent studies have characterized the changes in these receptors, as well as their endogenous ligands, in the basal ganglia in a variety of animal models of HD. The results are indicative that the endocannabinoid system becomes hypofunctional in this disease, which could be related to the hyperkinesia typical of the earliest phases of this disease. In addition, it has been proposed that the loss of these receptors might be involved in the process of pathogenesis itself. This, together with the well-known protective properties of cannabinoid-related compounds, suggest that, in addition to a symptomatic usefulness, cannabinoids might also serve to delay or to arrest the development of this disease. The present article will review all recent data dealing with the biochemical, pharmacological and therapeutic bases that support a potential role of cannabinoids in the pathogenesis and/or therapeutic treatment of this motor disorder.

Potential involvement of cannabinoid receptors in 3-nitropropionic acid toxicity in vivo

Several neurotransmitter systems are involved in the pathogenesis of Huntingtons disease. Here, we examined the involvement of cannabinoid CB1 receptors in striatal degeneration in the rat model of this disease generated by administration of 3-nitropropionic acid (3NP). Several days before onset of striatal degeneration, G-protein activation by cannabinoid agonists was significantly decreased whereas density and mRNA levels of CB1 receptors remained essentially normal. This change was transient, CB1 receptors recovering full functionality after few days. Later, at onset of striatal degeneration, profound alterations of CB1 receptors were detected, including marked reductions of their density, mRNA levels and coupling to G proteins. In these rats, the administration of the cannabinoid agonist Δ9-tetrahydrocannabinol was neuroprotective, which indicates that the early loss of CB1 receptor signaling could be instrumental in 3NP toxicity. In conclusion, the present study supports the hypothesis that cannabinoid receptors, possibly the CB1 receptor subtype, may be involved in HD pathogenesis and could be an interesting therapeutic target to slow disease progression.