Christopher Krewenka

Thymoquinone (TQ), an active compound from Nigella sativa seeds, is often described as a pharmacologically relevant compound with antioxidative properties, while the synthesis of TQ in the plant via oxidations makes it inapplicable for scavenging radicals. Therefore, the present study was designed to reassess the radical scavenging properties of TQ and explore a potential mode of action. The effects of TQ were studied in models with mitochondrial impairment and oxidative stress induced by rotenone in N18TG2 neuroblastoma cells and rotenone/MPP⁺ in primary mesencephalic cells. Tyrosine hydroxylase staining revealed that TQ significantly protected dopaminergic neurons and preserved their morphology under oxidative stress conditions. Quantification of the formation of superoxide radicals via electron paramagnetic resonance showed an initial increase in the level of superoxide radicals in the cell by TQ. Measurements in both cell culture systems revealed that the mitochondrial membrane potential was tendentially lowered, while ATP production was mostly unaffected. Additionally, the total ROS levels were unaltered. In mesencephalic cell culture under oxidative stress conditions, caspase-3 activity was decreased when TQ was administered. On the contrary, TQ itself tremendously increased the caspase-3 activity in the neuroblastoma cell line. Evaluation of the glutathione level revealed an increased level of total glutathione in both cell culture systems. Therefore, the enhanced resistance against oxidative stress in primary cell culture might be a consequence of a lowered caspase-3 activity combined with an increased pool of reduced glutathione. The described anti-cancer ability of TQ might be a result of the pro-apoptotic condition in neuroblastoma cells. Our study provides evidence that TQ has no direct scavenging effect on superoxide radicals.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder affecting 1% of the population above sixty years. It is caused by an interaction between genetic and environmental risk factors. Loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) is pathologically characterizing the disease and responsible for the cardinal motor symptoms, most notably, bradykinesia, rest tremors, rigidity, and loss of postural reflexes. Non-motor signs such as olfactory deficits, cognitive impairment, sleep behavior disorders, and gastrointestinal disturbances are reflecting disturbances in the non-dopaminergic system. They precede dopaminergic neuronal degenerations by 5–10 years and are considered the main contributors to patients’ disability, particularly after the successful implementation of levodopa (L-dopa) treatment of motor symptoms. The present general review aimed to briefly update non-motor signs and their underlying pathophysiology in PD.

Phytocannabinoids protect neurons against stressful conditions, possibly via the heme oxygenase (HO) system. In cultures of primary mesencephalic neurons and neuroblastoma cells, we determined the capability of cannabidiol (CBD) and tetrahydrocannabinol (THC) to counteract effects elicited by complex I-inhibitor rotenone by analyzing neuron viability, morphology, gene expression of IL6, CHOP, XBP1, HO-1 (stress response), and HO-2, and in vitro HO activity. Incubation with rotenone led to a moderate stress response but massive degeneration of dopaminergic neurons (DN) in primary mesencephalic cultures. Both phytocannabinoids inhibited in-vitro HO activity, with CBD being more potent. Inhibition of the enzyme reaction was not restricted to neuronal cells and occurred in a non-competitive manner. Although CBD itself decreased viability of the DNs (from 100 to 78%), in combination with rotenone, it moderately increased survival from 28.6 to 42.4%. When the heme degradation product bilirubin (BR) was added together with CBD, rotenone-mediated degeneration of DN was completely abolished, resulting in approximately the number of DN determined with CBD alone (77.5%). Using N18TG2 neuroblastoma cells, we explored the neuroprotective mechanism underlying the combined action of CBD and BR. CBD triggered the expression of HO-1 and other cell stress markers. Co-treatment with rotenone resulted in the super-induction of HO-1 and an increased in-vitro HO-activity. Co-application of BR completely mitigated the rotenone-induced stress response. Our findings indicate that CBD induces HO-1 and increases the cellular capacity to convert heme when stressful conditions are met. Our data further suggest that CBD via HO may confer full protection against (oxidative) stress when endogenous levels of BR are sufficiently high.

Phytocannabinoide sind Terpenphenole aus Cannabis spec. Sie binden im Gehirn u.a. an den CB1-Rezeptor, der beispielsweise auch an Membranen dopaminerger Neurone der Substantia nigra zu finden ist, welche bei Morbus Parkinson (MP) degenerieren. Ein Grund für den Zelltod ist die hohe Empfindlichkeit der dopaminergen Neurone gegenüber oxidativem Stress. In Zellkulturmodellen für MP wird er durch Komplex-I-Inhibitoren hervorgerufen, z.B. durch das Pestizid Rotenon. Da Phytocannabinoide auch antioxidative Eigenschaften aufweisen, wurde in unserer Studie untersucht, ob ein CB1-Rezeptoragonist, Tetrahydrocannabinol (THC), und ein Cannabinoid mit geringer Affinität zum CB1-Rezeptor, Cannabidiol (CBD), im Rotenon-Modell wirksam sind. Zur Darstellung dopaminerger Neurone mittels Anti-Tyrosinhydroxylase-Immunzytochemie wurden murine mesencephale Primärzellkulturen angelegt und am 12. Tag in vitro mit Kontrollmedium, Cannabinoid- (0,001–10µM), Rotenon- (80 nM) oder Cannabinoid/Rotenon-haltigem Medium für 48h inkubiert. Einer Verringerung der Anzahl der dopaminergen Neurone durch Rotenon (um 20%) konnte bei gleichzeitiger Behandlung mit THC und auch CBD entgegengewirkt werden (bei 10µM signifikant). Markanter war die Erhöhung der Glutathion-Spiegel, die bei 100 nM (THC) bzw. 10µM (CBD) signifikant war. In murinen Neuroblastomzellen wiesen Färbungen mit Propidiumiodid, einem Marker für Zelluntergang, ähnliche protektive Effekte beider Substanzen nach. Elektronenspinresonanzspektroskopische Untersuchungen zeigten eine Verdopplung der Superoxidradikalbildung in Kulturen mit Rotenon. Weder THC noch CBD wirkten dieser vermehrten Bildung entgegen. Im Rotenon-Modell für MP wird die schützende Wirkung von Phytocannabinoiden durch Stärkung der endogenen antioxidativen Mechanismen der Zellen vermittelt und nicht durch die Wirkung als Radikalfänger. Da auch CBD wirksam ist, ist die Erhöhung der Glutathion-Spiegel zumindest in Teilen nicht rezeptorvermittelt. Danksagung: Dieses Projekt ist eine Zusammenarbeit mit der Bionorica Research, Innsbruck, und wird vom FFG gefördert (Bridge 829646).