The growth/differentiation factor-15, GDF-15, has been found to be secreted by Schwann cells in the lesioned peripheral nervous system. To investigate whether GDF-15 plays a role in peripheral nerve regeneration, we substituted exogenous GDF-15 into 10-mm sciatic nerve gaps in adult rats and compared functional and morphological regeneration to a vehicle control group. Over a period of 11 weeks, multiple functional assessments, including evaluation of pinch reflexes, the Static Sciatic Index and of electrophysiological parameters, were performed. Regenerated nerves were then morphometrically analyzed for the number and quality of regenerated myelinated axons. Substitution of GDF-15 significantly accelerated sensory recovery while the effects on motor recovery were less strong. Although the number of regenerated myelinated axons was significantly reduced after GDF-15 treatment, the regenerated axons displayed advanced maturation corroborating the results of the functional assessments. Our results suggest that GDF-15 is involved in the complex orchestration of peripheral nerve regeneration after lesion.

The endocannabinoid system (ECS) consists particularly of cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, and enzymes that synthesize and degrade their ligands. It acts in a variety of organs and disease states ranging from cancer progression over neuropathic pain to neurodegeneration. Protein components engaged in the signaling, trafficking, and homeostasis machinery of the G-protein coupled CB2, are however largely unknown. It is therefore important to identify further interaction partners to better understand CB2 receptor functions in physiology and pathophysiology. For this purpose, we used an affinity purification and mass spectrometry-based proteomics approach of Strep-HA-CB2 receptor in HEK293 cells. After subtraction of background interactions and protein frequency library assessment we could identify 83 proteins that were classified by the identification of minimally 2 unique peptides as highly probable interactors. A functional protein association network analysis obtained an interaction network with a significant enrichment of proteins functionally involved in protein metabolic process, in endoplasmic reticulum, response to stress but also in lipid metabolism and membrane organization. The network especially contains proteins involved in biosynthesis and trafficking like calnexin, Sec61A, tubulin chains TUBA1C and TUBB2B, TMED2, and TMED10. Six proteins that were only expressed in stable CB2 expressing cells were DHC24, DHRS7, GGT7, HECD3, KIAA2013, and PLS1. To exemplify the validity of our approach, we chose a candidate having a relatively low number of edges in the network to increase the likelihood of a direct protein interaction with CB2 and focused on the scaffold/phagosomal protein p62/SQSTM1. Indeed, we independently confirmed the interaction by co-immunoprecipitation and immunocytochemical colocalization studies. 3D reconstruction of confocal images furthermore showed CB2 localization in close Frontiers in Molecular Neuroscience | www.frontiersin.org 1 September 2019 | Volume 12 | Article 224 Sharaf et al. Cannabinoid Receptor CB2 Interactomeproximity to p62 positive vesicles at the cell membrane. In summary, we provide a comprehensive repository of the CB2 interactome in HEK293 cells identified by a systematic unbiased approach, which can be used in future experiments to decipher the signaling and trafficking complex of this cannabinoid receptor. Future studies will have to analyze the exact mechanism of the p62-CB2 interaction as well as its putative role in disease pathophysiology.

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.

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.