Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

Autotaxin is primarily known for the formation of lysophosphatidic acid (LPA) from lysophosphatidylcholine. LPA is an important signaling phospholipid that can bind to six G protein–coupled receptors (LPA_1–6). The ATX-LPA signaling axis is a critical component in many physiological and pathophysiological conditions. Here, we describe a potent inhibition of Δ⁹-trans-tetrahydrocannabinol (THC), the main psychoactive compound of medicinal cannabis and related cannabinoids, on the catalysis of two isoforms of ATX with nanomolar apparent EC₅₀values. Furthermore, we decipher the binding interface of ATX to THC, and its derivative 9(R)-Δ6a,10a-THC (6a10aTHC), by X-ray crystallography. Cellular experiments confirm this inhibitory effect, revealing a significant reduction of internalized LPA₁in the presence of THC with simultaneous ATX and lysophosphatidylcholine stimulation. Our results establish a functional interaction of THC with autotaxin–LPA signaling and highlight novel aspects of medicinal cannabis therapy.

The functional importance of neuronal differentiation of the transmembrane proteins’ plasticity-related genes 3 (PRG3) and 5 (PRG5) has been shown. Although their sequence is closely related, they promote different morphological changes in neurons. PRG3 was shown to promote neuritogenesis in primary neurons; PRG5 contributes to spine induction in immature neurons and the regulation of spine density and morphology in mature neurons. Both exhibit intracellularly located C-termini of less than 50 amino acids. Varying C-termini suggested that these domains shape neuronal morphology differently. We generated mutant EGFP-fusion proteins in which the C-termini were either swapped between PRG3 and PRG5, deleted, or fused to another family member, plasticity-related gene 4 (PRG4), that was recently shown to be expressed in different brain regions. We subsequently analyzed the influence of overexpression in immature neurons. Our results point to a critical role of the PRG3 and PRG5 C-termini in shaping early neuronal morphology. However, the results suggest that the C-terminus alone might not be sufficient for promoting the morphological effects induced by PRG3 and PRG5.

Over the past few decades, many current uses for cannabinoids have been described, ranging from controlling epilepsy to neuropathic pain and anxiety treatment. Medicines containing cannabinoids have been approved by both the FDA and the EMA for the control of specific diseases for which there are few alternatives. However, the molecular-level mechanism of action of cannabinoids is still poorly understood. Recently, cannabinoids have been shown to interact with autotaxin (ATX), a secreted lysophospholipase D enzyme responsible for catalyzing lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a pleiotropic growth factor that interacts with LPA receptors. In addition, a high-resolution structure of ATX in complex with THC has recently been published, accompanied by biochemical studies investigating this interaction. Due to their LPA-like structure, endocannabinoids have been shown to interact with ATX in a less potent manner. This finding opens new areas of research regarding cannabinoids and endocannabinoids, as it could establish the effect of these compounds at the molecular level, particularly in relation to inflammation, which cannot be explained by the interaction with CB1 and CB2 receptors alone. Further research is needed to elucidate the mechanism behind the interaction between cannabinoids and endocannabinoids in humans and to fully explore the therapeutic potential of such approaches.

Autotaxin (ATX) is an enzyme primarily known for the production of lysophosphatidic acid. Being involved in the development of major human diseases, such as cancer and neurodegenerative diseases, the enzyme has been featured in multiple studies as a pharmacological target. We previously found that the cannabinoid tetrahydrocannabinol (THC) could bind and act as an excellent inhibitor of ATX. This study aims to use the cannabinoid scaffold as a starting point to find cannabinoid-unrelated ATX inhibitors, following a funnel down approach in which large chemical libraries sharing chemical similarities with THC were screened to identify lead scaffold types for optimization. This approach allowed us to identify compounds bearing chromone and indole scaffolds as promising ATX inhibitors. Further optimization led to MEY-003, which is characterized by the direct linkage of an N-pentyl indole to the 5,7-dihydroxychromone moiety. This molecule has potent inhibitory activity towards ATX-β and ATX-ɣ as evidenced by enzymatic studies and its mode of action was rationalized by structural biology studies.

Objective: This study was undertaken to characterize the functional impact of novel SLC12A5 variants in two unrelated patients with early onset developmental and epileptic encephalopathy (DEE) and to investigate the mechanisms underlying KCC2 dysfunction. Methods: Clinical, genetic, and functional analyses were performed in two patients (Cases A and B) with DEE. SLC12A5 encodes two KCC2 splice isoforms (KCC2a and KCC2b). Functional effects of the identified variants on KCC2b ion transport, phosphorylation, mRNA processing, and KCC2‐dependent synaptogenesis were assessed using in vitro assays in heterologous expression systems and primary neurons, supported by in silico structural modeling. Results: Both patients developed severe neonatal onset DEE characterized by developmental delay, axial hypotonia, extrapyramidal features, and bilateral migratory seizures within 24 h of birth. Both cases resulted in early mortality (Case A at 9 years; Case B at 6 months). Sequencing revealed distinct biallelic compound heterozygous SLC12A5 variants in both individuals, each inherited from one unaffected parent. Functional analyses demonstrated that in Case A, one variant markedly reduced KCC2‐mediated ion transport, whereas the second variant preserved transport activity but exhibited an altered phosphorylation profile at Ser940, located on the intracellular C ‐terminal region. This variant also disrupted wild‐type (WT) KCC2‐dependent excitatory synapse formation in immature rat hippocampal neurons. In Case B, one variant disrupted normal mRNA transcript processing consistent with loss of expression, and the second variant exhibited reduced ion transport activity. Significance: These data demonstrate that SLC12A5 ‐related DEE can result from combined impairment of KCC2‐dependent chloride homeostasis and disruption of chloride‐independent KCC2 functions critical for early neuronal development. This work expands the mutational and mechanistic spectrum of SLC12A5 ‐DEE and highlights the importance of KCC2 regulatory roles in early brain development, providing new knowledge and tools for basic research and potential avenues for targeted precision therapies.