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Cannflavins A and B are flavonoids that accumulate in the Cannabis sativa plant. These specialized metabolites are uniquely prenylated and highly lipophilic, which, a priori, may permit their interaction with membrane-bound enzymes and receptors. Although previous studies found that cannflavins can produce anti-inflammatory responses by inhibiting the biosynthesis of pro- inflammatory mediators, the full extent of their cellular influence remains to be understood. Here, we studied these flavonoids in relation to the Tropomyosin receptor kinase B (TrkB), a receptor tyrosine kinase that is activated by the growth factor brain-derived neurotrophic factor (BDNF). Using mouse primary cortical neurons, we first collected evidence that cannflavins prevent the accumulation of Activity-regulated cytoskeleton-associated (Arc, also known as Arg3.1) protein upon TrkB stimulation by exogenous BDNF in these cells. Consistent with this effect, we also observed a reduced activation of TrkB and downstream signaling effectors that mediate Arc mRNA transcription when BDNF was co-applied with the cannflavins. Of note, we also performed a high-throughput screen that demonstrated a lack of agonist action of cannflavins towards 320 different G protein-coupled receptors, a result that specifically limit the possibility of a TrkB transinactivation scenario via G protein signaling to explain our results with dissociated neurons. Finally, we used Neuro2a cells overexpressing TrkB to show that cannflavins can block the growth of neurites and increased survival rate produced by the higher abundance of the receptor in this model. Taken together, our study offers a new path to understand the reported effects of cannflavins and other closely related compounds in different cellular contexts.

Activity-regulated cytoskeleton-associated protein (Arc) is an immediate-early gene product that support neuroplastic changes important for cognitive function and memory formation. As a protein with homology to the retroviral Gag protein, a particular characteristic of Arc is its capacity to self-assemble into virus-like capsids that can package mRNAs and transfer those transcripts to other cells. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are coordinately regulated both temporally and spatially in neurons. The answer to this question we hypothesized must involve the occurrence of different protein post-translational modifications acting to confer specificity. In this study, we used mass spectrometry and sequence prediction strategies to map novel Arc phosphorylation sites. Our approach led us to recognize serine 67 (S67) and threonine 278 (T278) as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons that shares many functional overlaps with Arc and has, along with its interacting proteins such as the NMDA receptor, been implicated as a risk factor for psychiatric disorders. Furthermore, characterization of each residue using site-directed mutagenesis to create S67 and T278 mutant variants revealed that TNIK action at those amino acids can strongly influence Arc’s subcellular distribution and self-assembly as capsids. Together, our findings reveal an unsuspected connection between Arc and TNIK. Better understanding of the interplay between these two proteins in neuronal cells could lead to new insights about apparition and progression of psychiatric disorders.