Symptom-relieving and neuroprotective effects of the phytocannabinoid Δ⁹-THCV in animal models of Parkinson's disease

BACKGROUND AND PURPOSE

Previous findings have indicated that a cannabinoid, such as Δ⁹-THCV, which has antioxidant properties and the ability to activate CB₂ receptors but to block CB₁, might be a promising therapy for alleviating symptoms and delaying neurodegeneration in Parkinson''s disease (PD).

EXPERIMENTAL APPROACH

The ability of Δ⁹-THCV to reduce motor inhibition and provide neuroprotection was investigated in rats lesioned with 6-hydroxydopamine and in mice lesioned with lipopolysaccharide (LPS).

KEY RESULTS

Acute administration of Δ⁹-THCV attenuated the motor inhibition caused by 6-hydroxydopamine, presumably through changes in glutamatergic transmission. Moreover, chronic administration of Δ⁹-THCV attenuated the loss of tyrosine hydroxylase–positive neurones caused by 6-hydroxydopamine in the substantia nigra, through an effect related to its antioxidant properties (it was reproduced by cannabidiol -enriched botanical extract). In addition, CB₂ receptor–deficient mice responded to 6-hydroxydopamine in a similar manner to wild-type animals, and CB₂ receptors were poorly up-regulated in the rat substantia nigra in response to 6-hydroxydopamine. By contrast, the substantia nigra of mice that had been injected with LPS exhibited a greater up-regulation of CB₂ receptors. In these animals, Δ⁹-THCV also caused preservation of tyrosine hydroxylase–positive neurones. This effect probably involved CB₂ receptors as it was also elicited by the selective CB₂ receptor agonist, HU-308, and CB₂ receptor–deficient mice were more vulnerable to LPS lesions.

CONCLUSIONS AND IMPLICATIONS

Given its antioxidant properties and its ability to activate CB₂ but to block CB₁ receptors, Δ⁹-THCV has a promising pharmacological profile for delaying disease progression in PD and also for ameliorating parkinsonian symptoms.

LINKED ARTICLES

This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Apigenin induces DNA damage through the PKCδ-dependent activation of ATM and H2AX causing down-regulation of genes involved in cell cycle control and DNA repair

Apigenin, an abundant plant flavonoid, exhibits anti-proliferative and anti-carcinogenic activities through mechanisms yet not fully defined. In the present study, we show that the treatment of leukemia cells with apigenin resulted in the induction of DNA damage preceding the activation of the apoptotic program. Apigenin-induced DNA damage was mediated by p38 and protein kinase C-delta (PKCδ), yet was independent of reactive oxygen species or caspase activity. Treatment of monocytic leukemia cells with apigenin induced the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and histone H2AX, two key regulators of the DNA damage response, without affecting the ataxia-telangiectasia mutated and Rad-3-related (ATR) kinase. Silencing and pharmacological inhibition of PKCδ abrogated ATM and H2AX phosphorylation, whereas inhibition of p38 reduced H2AX phosphorylation independently of ATM. We established that apigenin delayed cell cycle progression at G1/S and increased the number of apoptotic cells. In addition, genome-wide mRNA analyses showed that apigenin-induced DNA damage led to down-regulation of genes involved in cell-cycle control and DNA repair. Taken together, the present results show that the PKCδ-dependent activation of ATM and H2AX define the signaling networks responsible for the regulation of DNA damage promoting genome-wide mRNA alterations that result in cell cycle arrest, hence contributing to the anti-carcinogenic activities of this flavonoid.     

How reliable is the behavioural evaluation of dyskinesia in animal models of Parkinson's disease?

In spite of the current availability of several pharmacological therapies for the treatment of Parkinson's disease, side effects are invariably manifested during long-term treatment. Dyskinesia, wearing-off and on-off are among the most disabling side effects produced by the dopamine precursor L-dihydroxyphenylalanine and, to a lesser degree, by other pharmacological treatments based on dopamine receptor agonism. Evaluation of the side effects, in particular dyskinesia, produced by antiparkinsonian drug treatments, therefore represents a critical issue in drug validation prior to a clinical trial. Moreover, a reliable model of dyskinesia is a fundamental requirement for the study of the as yet unknown mechanisms at the basis of this severely disabling side effect. The present review aims to provide a critical evaluation of the validity, reliability and utility of animal models of dyskinesia. In the first part of this review, we present a brief overview of the different models of Parkinson's disease focusing on those utilized for the evaluation of dyskinetic movements, then proceed to critically examine the turning behaviour model in an attempt to assess the way in which it has influenced the evaluation of drugs utilized in the treatment of Parkinson's disease. Subsequently, the various models of dyskinesia are reviewed and conclusions are drawn as to how the environment in which experiments are performed can influence the behaviour observed.     

γ-Tocotrienol induces apoptosis in human T cell lymphoma through activation of both intrinsic and extrinsic pathways

Tocotrienols are members of vitamin E family and possess broad biological activities including antioxidant, anti-inflammatory and antitumor effects. In the present study, we examine the potential of α-tocotrienol (AT) and γ-tocotrienol (GT) in inhibiting the proliferation of human T cell lymphoma Jurkat cells and elucidate the pathways involved in anti tumor effects of GT. GT but not AT inhibited proliferation and induced apoptosis in Jurkat cells in a dose dependent manner. GT treatment resulted in elevated mitochondrial ROS production, activation of JNK and suppression of ERK and p38 MAPK. GT also induced calcium release, loss of mitochondrial membrane potential and cytochrome c release from the mitochondria. These changes were accompanied by increase in Bax expression with a concomitant decrease in Bcl-xl expression suggesting activation of mitochondrial apoptotic pathway. GT induced increase in mitochondrial ROS was abrogated by catalase. Besides, GT also up-regulated surface expression of Fas and FasL on Jurkat cells. Further, caspase activation and PARP degradation were also seen in cells treated with GT. Inhibitors of caspase-8 and caspase-9 significantly abrogated GT mediated apoptosis. In contrast GT was not toxic to normal human peripheral blood mononuclear cells suggesting differential cytotoxicity towards normal lymphocytes and transformed lymphoma cells. Cellular uptake studies with tocotrienols showed higher intracellular accumulation of GT as compared to AT which may be responsible for its better antitumor activity. Our results show antitumor effects of GT in human lymphoma cells via increased mitochondrial ROS generation and activation of both intrinsic and extrinsic apoptotic pathways.     

A prolyl oligopeptidase inhibitor, KYP-2047, reduces α-synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease

BACKGROUND AND PURPOSE

The aggregation of α-synuclein is connected to the pathology of Parkinson''s disease and prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on α-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human α-syn and in the brains of two A30P α-synuclein transgenic mouse strains.

EXPERIMENTAL APPROACH

Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild-type or transgenic mice were treated for 5 days with KYP-2047 (2 × 3 mg·kg⁻¹ a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble α-synuclein protein levels were measured by Western blot. α-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and α-synuclein,and the effect of KYP-2047 on cell viability were also investigated.

KEY RESULTS

In cell lines, oxidative stress induced a robust aggregation of α-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with α-synuclein inclusions while abolishing the colocalization of α-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated α-synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5-day treatment with the PREP inhibitor reduced the amount of α-synuclein immunoreactivity and soluble α-synuclein protein in the brain.

CONCLUSIONS AND IMPLICATIONS

The results suggest that the PREP may play a role in brain accumulation and aggregation of α-synuclein, while KYP-2047 seems to effectively prevent these processes.     

Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?

Free radicals and other reactive species (RS) are thought to play an important role in many human diseases. Establishing their precise role requires the ability to measure them and the oxidative damage that they cause. This article first reviews what is meant by the terms free radical, RS, antioxidant, oxidative damage and oxidative stress. It then critically examines methods used to trap RS, including spin trapping and aromatic hydroxylation, with a particular emphasis on those methods applicable to human studies. Methods used to measure oxidative damage to DNA, lipids and proteins and methods used to detect RS in cell culture, especially the various fluorescent "probes" of RS, are also critically reviewed. The emphasis throughout is on the caution that is needed in applying these methods in view of possible errors and artifacts in interpreting the results.     

Tat-DJ-1 inhibits oxidative stress-mediated RINm5F cell death through suppression of NF-κB and MAPK activation

Oxidative stress is highly involved in the development of diabetes mellitus by destruction of pancreatic β-cells. DJ-1 is an antioxidant protein and DJ-1 expression levels are known to be reduced in diabetes mellitus. Thus, we examined the effects of DJ-1 protein against oxidative stress-induced pancreatic β-cell (RINm5F) death using cell permeable wild-type and mutant-type (C106A) Tat-DJ-1 proteins, which both efficiently transduced into RINm5F cells. Intracellular stability of wild-type Tat-DJ-1 persisted two times longer than C106A Tat-DJ-1. Wild-type Tat-DJ-1 protein markedly protected cells from hydrogen peroxide-induced toxicities such as cell death, reactive oxygen species generation, and DNA fragmentation. Further, wild-type Tat-DJ-1 protein significantly inhibited hydrogen peroxide-induced activation of mitogen-activated protein kinases and NF-κB signaling. On the other hand, C106A Tat-DJ-1 protein did not show the same protective effects. These results indicate that wild-type Tat-DJ-1 inhibits oxidative stress-induced cellular toxicity and activation of mitogen-activated protein kinases and NF-κB signals in RINm5F cells. These results suggest that wild-type Tat-DJ-1 protein may be a potential therapeutic agent against diabetes mellitus or toward the prevention of pancreatic β-cell destruction.     

Differential susceptibility to the PPAR-γ agonist pioglitazone in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine rodent models of Parkinson's disease

A growing body of evidence suggests that peroxisome proliferator-activated receptor (PPAR) agonists are valuable candidates as disease modifiers in Parkinson's disease (PD) and may thus enable neuroprotection and preserve motor function. The present study sought to evaluate the effect of the PPAR-gamma agonist pioglitazone in two different animal models of PD. The study was based on nigral dopaminergic neuron labelling and the assessment of motor behaviour in (i) the frequently investigated MPTP mouse model and (ii) the less well-known bilateral 6-OHDA rat model. In MPTP-injected mice, pioglitazone reversed body weight loss and the reduction in rearing frequency and induced significant neuroprotection of the nigrostriatal dopaminergic pathway (by 24%, compared with vehicle). In contrast, pioglitazone did not have any effect on the 73.5% loss of dopaminergic neurons or motor impairments (a reduced rearing frequency and a loss of strength in the forepaws) in bilateral 6-OHDA rats. The PPAR-gamma agonist pioglitazone had a significant neuroprotective effect in MPTP mice but not in bilateral 6-OHDA rats. The various effects of PPAR agonists in both models can be accounted by the different action mechanism of the 2 toxins or by the fact that 3μg 6-OHDA injection was too harmful to be alleviated by the compound. This work supports PPAR-agonists to be relevant in the therapeutic strategy research in Parkinson's disease and highlights the importance in evaluating neuroprotective agent in different models.     

Apremilast prevent doxorubicin-induced apoptosis and inflammation in heart through inhibition of oxidative stress mediated activation of NF-κB signaling pathways

Background

Doxorubicin is an effective, potent and commonly used anthracycline-related anticancer drug; however, cardiotoxicity compromises its therapeutic potential. Apremilast, a novel phosphodiesterase type 4-inhibitor, reported to have anti-inflammatory effects and modulating many inflammatory mediators.

5-HT induces temporomandibular joint nociception in rats through the local release of inflammatory mediators and activation of local β adrenoceptors

Objectives

The present studies assessed the effects of adolescent and adult ethanol exposure on the rewarding effects of cocaine as measured with the conditioned place preference procedure.

Methods

Male rats were exposed to intraperitoneal (IP) injections of ethanol or vehicle for 10 days [postnatal days (PNDs) 30-39 or PNDs 70-79; 2 mg/kg]. Place preference conditioning began on PND 65 or PND 105, respectively, and consisted of a baseline test followed by four conditioning cycles with either 0, 5, 10 or 20 mg/kg cocaine. Following the fourth conditioning cycle a final preference test was performed. Changes in time on the drug-paired side between the baseline and final test were analyzed.

Results

Animals exposed to vehicle (during adolescence or adulthood) showed a significant place preference at 20 mg/kg cocaine. Animals exposed to ethanol (during adolescence or adulthood) showed a significant place preference at 10 mg/kg cocaine.

Conclusions

Exposure to ethanol (adolescents or adults) sensitized the rewarding effects of cocaine. This may indicate an increase in the abuse liability of cocaine following a history of ethanol exposure.     

Cigarette smoke extract induces activation of β-catenin/TCF signaling through inhibiting GSK3β in human alveolar epithelial cell line

Cigarette smoke is known to have various injurious and cytotoxic effects on alveolar epithelial cells. However, the mechanism about the effects caused by cigarette smoke on alveolar epithelial cells remains unclear.     

Acetyl-L-carnitine and α-lipoic acid affect rotenone-induced damage in nigral dopaminergic neurons of rat brain, implication for Parkinson's disease therapy

Although the mechanisms of neurodegeneration in Parkinson's disease are not fully understood, mitochondrial dysfunction, oxidative stress and environmental toxins may be involved. The current research was directed to investigate the protective role of two bioenergetic antioxidants, acetyl-l-carnitine and α-lipoic acid, in rotenone-parkinsonian rats. Ninety six male rats were divided into five groups. Group I is the vehicle-injected group, group II is the disease control group and was injected with six doses of rotenone (1.5 mg/kg/48 h, s.c.). Groups III, IV and V received rotenone in addition to acetyl-l-carnitine (100 mg/kg/day, p.o.), α-lipoic acid (50 mg/kg/day, p.o.) or their combination, respectively. Results showed that rotenone-treated rats exhibited bradykinesia and motor impairment in the open-field and square bridge tests. In addition, ATP level was decreased whereas lipid peroxides and protein carbonyls increased in the striata of rotenone-treated rats as compared to vehicle-treated rats. Treatment with acetyl-l-carnitine or α-lipoic acid improved the motor performance and reduced the level of lipid peroxides in rat brains as compared to rotenone group. Further, ATP production was enhanced along with acetyl-l-carnitine treatments (p ≤ 0.05). Taken together, our study reinforces the view that acetyl-l-carnitine and α-lipoic acid are promising candidates for neuroprotection in Parkinson's disease.     

Tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury in different models possibly through suppression of NF-κB activation

Tylvalosin, a new broad-spectrum, third-generation macrolides, may exert a variety of pharmacological activities. Here, we report on its anti-oxidative and anti-inflammatory activity in RAW 264.7 macrophages and mouse treated with lipopolysaccharide (LPS) as well as piglet challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Tylvalosin treatment markedly decreased IL-8, IL-6, IL-1β, PGE2, TNF-α and NO levels in vitro and in vivo. LPS and PRRSV-induced reactive oxygen species (ROS) production, and the lipid peroxidation in mice lung tissues reduced after tylvalosin treatments. In mouse acute lung injury model induced by LPS, tylvalosin administration significantly attenuated tissues injury, and reduced the inflammatory cells recruitment and activation. The evaluated phospholipase A2 (PLA2) activity and the increased expressions of cPLA2-IVA, p-cPLA2-IVA and sPLA2-IVE were lowered by tylvalosin. Consistent with the mouse results, tylvalosin pretreatment attenuated piglet lung scores with improved growth performance and normal rectal temperature in piglet model induced by PRRSV. Furthermore, tylvalosin attenuated the IκBα phosphorylation and degradation, and blocked the NF-κB p65 translocation. These results indicate that in addition to its direct antimicrobial effect, tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury through suppression of NF-κB activation.     

β-Adrenergic receptors stimulate interleukin-6 production through Epac-dependent activation of PKCδ/p38 MAPK signalling in neonatal mouse cardiac fibroblasts

BACKGROUND AND PURPOSE IL-6 plays crucial roles in cardiac hypertrophy, cardiac fibrosis and heart failure. Activation of β-adrenoceptors induced IL-6 production in neonatal mouse cardiac fibroblasts (NMCFs) through a G(s) /adenylate cyclase/cAMP/p38 MAPK pathway but independent of PKA. However, how cAMP activates p38 MAPK is still not defined. In this study, we have assessed the role of the exchange protein directly activated by cAMP (Epac) and PKCδ in p38 MAPK activation and IL-6 production by stimulated by the β-adrenoceptor agonist isoprenaline in NMCFs. EXPERIMENTAL APPROACH The IL-6 concentration in cell culture supernatants was measured by ELISA. The levels of phosphorylated and total p38 MAPK and PKCδ were determined by Western blot analysis. The translocation of PKCδ was determined by immunoblotting the soluble and particulate fractions. Expression of Epac1 or PKCδ was knocked down by the corresponding, adenovirus-mediated, small hairpin RNA (shRNA). RESULTS In NMCFs, activation of β-adrenoceptors enhanced PKCδ phosphorylation and translocation. Furthermore, knock-down of the PKCδ isoform using an adenovirus-mediated shRNA markedly down-regulated IL-6 induction by NMCFs stimulated with isoprenaline. Moreover, knock-down of Epac1 confirmed that Epac1 was upstream of PKCδ in IL-6 production. Additionally, both Epac1 and PKCδ mediated the p38 MAPK activation induced by isoprenaline. CONCLUSIONS AND IMPLICATIONS β-Adrenoceptor agonists activate a cAMP/Epac/PKCδ/p38 MAPK pathway to produce IL-6 in NMCFs. This study identifies Epac as the link between cAMP and p38 MAPK signalling pathways and demonstrates that PKCδ can function as a novel downstream effector of this β-adrenoceptor/cAMP/Epac pathway.     

Dibenzofuran induces oxidative stress,disruption of trans-mitochondrial membrane potential (ΔΨm) and G1 arrest in human hepatoma cell line

Dioxins are a class of extremely toxic environmentally persistent pollutant, comprised of halogenated dibenzo-p-dioxins, dibenzofurans and biphenyls. Despite significant human exposure via multiple routes, very little is known about toxicity induced by dibenzofuran (DF). Current study shed lights on the potential toxicity mechanism of DF using human hepatoma cell line (HepG2). It was observed that the exposure to DF potentiate oxidative stress, apoptosis and necrosis at 10 μM within 8 h in HepG2 cells. Interestingly, when we pre-incubated the cells with α-NF (1 nM) for 12 h, an aromatic hydrocarbon receptor antagonist, the IC₅₀ of DF increased by 14 folds indicating the cytoprotective ability of α-NF from DF induced toxicity. Furthermore, three additional metabolites were observed while studying the metabolic profile of DF in HepG2 cells with and without pre-incubation with α-NF using chromatography–mass spectroscopy (GC–MS). Of these, two metabolites were characterized as dihydroxylated derivative of DF and third metabolite was characterized as quinone derivative of DF. By flow cytometry and confocal laser microscopy analysis we followed the ROS formation after DF (10 μM) exposure for 3 h. Significantly low ROS was generated in cells which were pre-incubated with α-NF than cells which were not pre-incubated with α-NF underlining the importance of metabolism in DF toxicity. The same pattern of protection was consistent while measuring mitochondrial membrane potential (MMP), i.e., less MMP dip was observed in ‘with α-NF pre-incubated and DF (10 μM) exposed cells’ than ‘without α-NF pre-incubated but DF exposed cells’. In cell cycle studies, it was confirmed that cell population of HepG2 at G1 stage progressively increased in number (∼74%) within 24 h. Thus, DF and its metabolites induce significantly higher cytotoxicity after metabolism in HepG2 cells than its parent compound (DF) by ROS formation, MMP dip and impaired cell cycle.     

Corynoxine B targets at HMGB1/2 to enhance autophagy for α-synuclein clearance in fly and rodent models of Parkinson's disease

Parkinson’s disease(PD) is the most common neurodegenerative movement disease. It is featured by abnormal alphα-synuclein(α-syn) aggregation in dopaminergic neurons in the substantia nigra. Macroautophagy(autophagy) is an evolutionarily conserved cellular process for degradation of cellular contents, including protein aggregates, to maintain cellular homeostasis. Corynoxine B(Cory B), a natural alkaloid isolated from Uncaria rhynchophylla(Miq.) Jacks., has been reported to promote the clearance ...     

Modulation of indole ring annulation in ergoline template: chemistry,receptor binding and in vivo pharmacology with 6-OHDA model of Parkinson’s disease

l-dopa provides symptomatic cure in Parkinson’s disease (PD); however, long-term treatment leads to the development of motor fluctuations and dyskinesia. l-dopa-sparing monotherapy involves treatment with dopamine agonists (DAs) exerting their action through dopamine receptor, which delays l-dopa treatment in PD. Cis and trans N-4-substituted-2,3,4,4a,5,6,11,11a-octahydro-1H-pyrido[3,2-a] carbazoles were synthesized by modulating the indole ring annulations pattern of ergoline skeleton to identify novel DAs with promising antiparkinsonian activity. The compounds 16 and 20 with the Ki value of 1.31 and 1.09 nM, respectively, exhibiting D2-like activity were selected for evaluation of antiparkinsonian activity in 6-OHDA-induced unilateral lesioned rat model. Persuasively, both compounds 16 and 20 showed antiparkinsonian activity in a dose-dependant manner and slightly longer duration of action as compared to apomorphine.     

Fibroblast growth factor-20 protects against dopamine neuron loss in vitro and provides functional protection in the 6-hydroxydopamine-lesioned rat model of Parkinson's disease

Fibroblast growth factor-20 (FGF-20) has been shown to protect dopaminergic neurons against a range of toxic insults in vitro, through activation of fibroblast growth factor receptor 1 (FGFR1). This study set out to examine whether FGF-20 also displayed protective efficacy in the unilateral, 6-hydroxydopamine (6-OHDA) lesion rat model of Parkinson's disease. Initial studies demonstrated that, in embryonic ventral mesencephalic (VM) cultures, FGFR1 was expressed on tyrosine hydroxylase (TH)-positive neurons and that, in line with previous data, FGF-20 (100 and 500 ng/ml) almost completely protected these TH-positive neurons against 6-OHDA-induced toxicity. Co-localisation of FGFR1 and TH staining was also demonstrated in the substantia nigra pars compacta (SNpc) of naïve adult rat brain. In animals subject to 6-OHDA lesion of the nigrostriatal tract, supra-nigral infusion of FGF-20 (2.5 μg/day) for 6 days post-lesion gave significant protection (～40%) against the loss of TH-positive cells in the SNpc and the loss of striatal TH immunoreactivity. This protection of the nigrostriatal tract was accompanied by a significant preservation of gross locomotion and fine motor movements and reversal of apomorphine-induced contraversive rotations, although forelimb akinesia, assessed using cylinder test reaching, was not improved. These results support a role for FGF-20 in preserving dopamine neuron integrity and some aspects of motor function in a rodent model of Parkinson's disease (PD) and imply a potential neuroprotective role for FGF-20 in this disease.