Untersuchung der differenziellen Genexpression in sensorischen und motorischen Nervenästen des peripheren Nervensystems

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Cannabinoids in Glioblastoma Therapy: New Applications for Old Drugs

Glioblastoma (GBM) is the most malignant brain tumor and one of the deadliest types of solid cancer overall. Despite aggressive therapeutic approaches consisting of maximum safe surgical resection and radio-chemotherapy, more than 95% of GBM patients die within 5 years after diagnosis. Thus, there is still an urgent need to develop novel therapeutic strategies against this disease. Accumulating evidence indicates that cannabinoids have potent anti-tumor functions and might be used successfully in the treatment of GBM. This review article summarizes the latest findings on the molecular effects of cannabinoids on GBM, both in vitro and in (pre-) clinical studies in animal models and patients. The therapeutic effect of cannabinoids is based on reduction of tumor growth via inhibition of tumor proliferation and angiogenesis but also via induction of tumor cell death. Additionally, cannabinoids were shown to inhibit the invasiveness and the stem cell-like properties of GBM tumors. Recent phase II clinical trials indicated positive results regarding the survival of GBM patients upon cannabinoid treatment. Taken together these findings underline the importance of elucidating the full pharmacological effectiveness and the molecular mechanisms of the cannabinoid system in GBM pathophysiology

Monitoring Cannabinoid CB2 -Receptor Mediated cAMP Dynamics by FRET-Based Live Cell Imaging

G-protein coupled cannabinoid CB2 receptor signaling and function is primarily mediated by its inhibitory effect on adenylate cyclase. The visualization and monitoring of agonist dependent dynamic 3′,5′-cyclic adenosine monophosphate (cAMP) signaling at the single cell level is still missing for CB2 receptors. This paper presents an application of a live cell imaging while using a Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, for quantification of cAMP. We established HEK293 cells stably co-expressing human CB2 and Epac1-camps and quantified cAMP responses upon Forskolin pre-stimulation, followed by treatment with the CB2 ligands JWH-133, HU308, β-caryophyllene, or 2-arachidonoylglycerol. We could identify cells showing either an agonist dependent CB2-response as expected, cells displaying no response, and cells with constitutive receptor activity. In Epac1-CB2-HEK293 responder cells, the terpenoid β-caryophyllene significantly modified the cAMP response through CB2. For all of the tested ligands, a relatively high proportion of cells with constitutively active CB2 receptors was identified. Our method enabled the visualization of intracellular dynamic cAMP responses to the stimuli at single cell level, providing insights into the nature of heterologous CB2 expression systems that contributes to the understanding of Gαi-mediated G-Protein coupled receptor (GPCR) signaling in living cells and opens up possibilities for future investigations of endogenous CB2 responses

Behavioral Studies of p62 KO Animals with Implications of a Modulated Function of the Endocannabinoid System

Elementary emotional states and memory can be regulated by the homeostasis of the endocannabinoid system (ECS). Links between the ECS and the autophagy receptor p62 have been found at the molecular level and in animal studies. This project aimed to validate the anxiety and memory phenotype of p62 knockout (KO) animals and whether the ECS plays a role in this. We examined the behavior of p62 KO animals and analyzed whether endocannabinoid levels are altered in the responsible brain areas. We discovered in age-dependent obese p62 KO mice decreased anandamide levels in the amygdala, a brain structure important for emotional responses. Against our expectation, p62 KO animals did not exhibit an anxiety phenotype, but showed slightly increased exploratory behavior as evidenced in novel object and further tests. In addition, KO animals exhibited decreased freezing responses in the fear conditioning. Administration of the phytocannabinoid delta9-tetrahydrocannabinol (THC) resulted in lesser effects on locomotion but in comparable hypothermic effects in p62 KO compared with WT littermates. Our results do not confirm previously published results, as our mouse line does not exhibit a drastic behavioral phenotype. Moreover, we identified further indications of a connection to the ECS and hence offer new perspectives for future investigations

Stable Adult Hippocampal Neurogenesis in Cannabinoid Receptor CB2 Deficient Mice

The G-protein coupled cannabinoid receptor 2 (CB2) has been implicated in the regulation of adult neurogenesis in the hippocampus. The contribution of CB2 towards basal levels of proliferation and the number of neural progenitors in the subgranular zone (SGZ) of the dentate gyrus, however, remain unclear. We stained hippocampal brain sections of 16- to 17-week-old wildtype and CB2-deficient mice, for neural progenitor and immature neuron markers doublecortin (DCX) and calretinin (CR) and for the proliferation marker Ki67 and quantified the number of positive cells in the SGZ. The quantification revealed that CB2 deficiency neither altered overall cell proliferation nor the size of the DCX+ or DCX and CR double-positive populations in the SGZ compared to control animals. The results indicate that CB2 might not contribute to basal levels of adult neurogenesis in four-month-old healthy mice. CB2 signaling might be more relevant in conditions where adult neurogenesis is dynamically regulated, such as neuroinflammation

Correction: Reelin controls the positioning of brainstem serotonergic raphe neurons.

[This corrects the article DOI: 10.1371/journal.pone.0200268.]

The cannabinoid receptor type 2 (CNR2) gene is associated with human osteoporosis

Abstract Genetic variants within the CNR2 gene encoding the cannabinoid receptor CB2 have been shown to be associated with osteoporosis and low bone mineral density (BMD) in case-control studies. We now examined the association of polymorphisms in CNR2 with hand bone strength in an ethnically homogeneous healthy family sample of European origin (Chuvashians) living in Russia. We show that non-synonymous CNR2 SNPs are signiWcantly associated with radiographic hand BMD and breaking bending resistance index (BBRI) by two diVerent transmission disequilibrium tests. For both tests highly signiWcant p values (ranging from 0.007 to 0.008 for hand BMD, and from 0.001 to 0.003 for BBRI) were also obtained with additional SNPs at the CNR2 locus. The associations remained signiWcant after correction for multiple testing. In conclusion, in addition to the association of CNR2 polymorphisms with low BMD at selected clinically relevant skeletal sites, we now report their signiWcant association with hand bone strength phenotypes using a family-based study design implying an even broader impact of genetic variation at the CNR2 locus on bone structure and function

Dynein Light Chain Protein Tctex1: A Novel Prognostic Marker and Molecular Mediator in Glioblastoma

The purpose of this study was to determine the role of Tctex1 (DYNLT1, dynein light chain-1) in the pathophysiology of glioblastoma (GBM). To this end, we performed immunohistochemical analyses on tissues from GBM patients (n = 202). Tctex1 was additionally overexpressed in two different GBM cell lines, which were then evaluated in regard to their proliferative and invasive properties. We found that Tctex1 levels were significantly higher in GBM compared to healthy adjacent brain tissues. Furthermore, high Tctex1 expression was significantly associated with the short overall- (p = 0.002, log-rank) and progression-free (p = 0.028, log-rank) survival of GBM patients and was an independent predictor of poor overall survival in multivariate Cox-regression models. In vitro, Tctex1 promoted the metabolic activity, anchorage-independent growth and proliferation of GBM cells. This phenomenon was previously shown to occur via the phosphorylation of retinoblastoma protein (phospho-RB). Here, we found a direct and significant correlation between the levels of Tctex1 and phospho-RB (Ser807/801) in tissues from GBM patients (p = 0.007, Rho = 0.284, Spearman’s rank). Finally, Tctex1 enhanced the invasiveness of GBM cells and the release of pro-invasive matrix metalloprotease 2 (MMP2). These findings indicate that Tctex1 promotes GBM progression and therefore might be a useful therapeutic target in this type of cancer

Reelin controls the positioning of brainstem serotonergic raphe neurons

<div><p>Serotonin (5-HT) acts as both a morphogenetic factor during early embryonic development and a neuromodulator of circuit plasticity in the mature brain. Dysregulation of serotonin signaling during critical periods is involved in developmental neurological disorders, such as schizophrenia and autism. In this study we focused on the consequences of defect <i>reelin</i> signaling for the development of the brainstem serotonergic raphe system. We observed that <i>reelin</i> signaling components are expressed by serotonergic neurons during the critical period of their lateral migration. Further, we found that <i>reelin</i> signaling is important for the normal migration of rostral, but not caudal hindbrain raphe nuclei and that <i>reelin</i> deficiency results in the malformation of the paramedian raphe nucleus and the lateral wings of the dorsal raphe nuclei. Additionally, we showed that serotonergic neurons projections to laminated brain structures were severely altered. With this study, we propose that the perturbation of canonical <i>reelin</i> signaling interferes with the orientation of tangentially, but not radially, migrating brainstem 5-HT neurons. Our results open the window for further studies on the interaction of <i>reelin</i> and serotonin and the pathogenesis of neurodevelopmental disorders.</p></div