The research, led by Shuhan Lv and Qian Chen and their colleagues, focused on Olfr544 for a compelling reason. Previous work had shown that this receptor is highly expressed in the cerebral cortex and hippocampus of nine-month-old 5×FAD transgenic mice, a widely used model of Alzheimer’s disease. Its expression localizes near amyloid-beta plaques, the pathological hallmark of the disease, and its mRNA levels fluctuate with both age and disease progression. That pattern alone would be intriguing, but Olfr544 also belongs to a small club of olfactory receptors that have been deorphanized, meaning scientists know their chemical activator. In this case, the trigger is azelaic acid, a naturally occurring dicarboxylic acid that has already been implicated in metabolic regulation elsewhere in the body, including glucagon secretion in pancreatic alpha cells, lipolysis, and mitochondrial biogenesis in skeletal muscle. What remained entirely unknown was the receptor’s function inside the nervous system.

To probe that function, the team turned to the mouse neuroblastoma cell line N2a, a standard workhorse for studying neuronal differentiation and neurite extension. Their first task was to confirm that these cells genuinely possess the machinery for Olfr544 signaling. Using reverse transcription polymerase chain reaction followed by agarose gel electrophoresis and DNA sequencing, they demonstrated that N2a cells endogenously express not just Olfr544 itself but also its canonical downstream partners, the olfactory G protein Gαolf and adenylyl cyclase 3. That trio constitutes the full signaling axis that olfactory receptors use in the nose, and its presence in neuronal cells suggested that Olfr544 could operate as a functional GPCR in the brain rather than sitting there as molecular decoration.

The functional experiments began with azelaic acid. After establishing with an MTT assay that concentrations up to 640 micromolar had no effect on N2a cell viability, the researchers treated serum-deprived cultures with 500 micromolar of the compound for 24 hours. Under phase-contrast microscopy, the effect was unmistakable. Roughly 30 percent of treated cells extended neurites, defined as processes longer than the cell body’s diameter, compared with only about 10 percent of serum-deprived controls. Because short-term serum deprivation itself modestly promotes neurite outgrowth, the azelaic acid effect represented a genuine, receptor-linked enhancement of neuronal differentiation. Direct morphological comparison with receptor-deficient cells proved technically awkward because Olfr544 knockout cells grow and adhere poorly even at baseline, so the team pivoted to a cleaner strategy: dissect the downstream signaling and transcriptional consequences of receptor loss.

Before those genetic experiments, the researchers sought independent confirmation that Olfr544 is sufficient, on its own, to drive CREB-dependent transcription. They cloned the full-length mouse Olfr544 cDNA into a specialized expression vector that promotes proper membrane trafficking of olfactory receptors, then co-transfected it into HEK293T cells, which lack endogenous Olfr544, together with a CREB-responsive dual-luciferase reporter. When azelaic acid was added, luciferase activity surged in receptor-expressing cells but stayed flat in empty-vector controls. Co-treatment with SQ22536, an inhibitor of adenylyl cyclase, markedly blunted the response, confirming that the signal flowed through cAMP production. This heterologous reconstitution ruled out the possibility that azelaic acid’s effects in neuronal cells depend on indirect pathways unique to the N2a environment, and it established a direct mechanistic line from receptor to transcription factor.

The genetic core of the study came from CRISPR-Cas9 editing. The team designed two single-guide RNAs targeting exon 2 of the mouse Olfr544 gene, transfected them into N2a cells, selected edited populations with puromycin, and isolated monoclonal lines by limiting dilution. Sanger sequencing confirmed a homozygous 149-base-pair deletion that introduces a frameshift and a premature stop codon, and reverse transcription PCR showed loss of the wild-type transcript, consistent with nonsense-mediated decay. The authors candidly note that no validated specific antibody existed to confirm receptor loss at the protein level, a limitation they flag explicitly. Still, the functional evidence was decisive. When wild-type N2a cells were stimulated with 50 micromolar azelaic acid for 30 minutes, levels of phosphorylated CREB, the transcription factor that sits at the crossroads of neuronal survival, plasticity, and memory formation, rose significantly. In knockout cells, that increase vanished entirely, while basal pCREB remained unchanged, proving that azelaic acid’s activation of CREB is strictly Olfr544-dependent.

The team then traced the pathway upstream of CREB. Canonical GPCR signaling proceeds through Gαs proteins to activate adenylyl cyclase, elevate intracellular cAMP, and stimulate protein kinase A, which in turn phosphorylates CREB. Enzyme-linked immunosorbent assays showed that azelaic acid triggered a rapid rise in intracellular cAMP in wild-type cells, though a smaller one than forskolin, a direct adenylyl cyclase activator, could produce. In knockout cells, the cAMP response to azelaic acid was completely abolished, and PKA activity followed the same pattern, rising with drug treatment in wild-type cells but not in receptor-deficient ones. Together, these measurements delineate a clean linear cascade: Olfr544 activation, cAMP elevation, PKA activation, CREB phosphorylation. It is the textbook GPCR framework, operating in a neuronal context and converging on a transcription factor that governs synaptic plasticity.

To see what genes this cascade controls, the researchers performed RNA sequencing on wild-type and Olfr544 knockout cells, generating more than 41 gigabytes of clean data with mapping rates between 86 and 95 percent. Differential expression analysis identified 1,727 genes whose expression changed, 1,397 upregulated and 330 downregulated. Enrichment analysis revealed a striking asymmetry. Upregulated genes clustered in the neuroactive ligand-receptor interaction pathway, involving 41 genes encoding GPCRs and ion channels, which the authors interpret as possible compensatory mechanisms. Downregulated genes were concentrated in the motor protein pathway, including members of the myosin, actin, and kinesin families. That downregulation is precisely what one would expect if Olfr544 supports neurite dynamics, because motor proteins are essential for the cytoskeletal remodeling that drives process extension and neuronal morphogenesis. The authors caution that the transcriptomic comparison used a single monoclonal knockout line, so the results should be read as hypothesis-generating candidate pathways rather than a definitive catalog of receptor targets.

From the downregulated genes, computational prediction of CREB binding sites flagged three high-confidence candidates: Igf2bp3, Sorbs2, and Kctd14. Independent validation in the unedited parental cell line strengthened the case, particularly for IGF2BP3, an mRNA-binding protein implicated in axon guidance and synaptic plasticity. Its protein level rose significantly with azelaic acid treatment in wild-type cells and was markedly reduced in knockout cells, with no rescue by the agonist, making it the most robust Olfr544-dependent effector identified. SORBS2, which maintains dendritic complexity and synaptic integrity, and KCTD14, a member of a family linked to neurodevelopmental disorders, showed consistent downward trends in knockout cells, though not every pairwise comparison reached statistical significance. The authors are careful to note that the link between CREB activation and these targets remains correlative and that chromatin immunoprecipitation studies will be needed to confirm direct transcriptional regulation.

The bigger picture is what makes this work resonate beyond the cell culture hood. Olfactory receptors account for roughly 60 percent of human GPCRs, and mounting evidence ties their brain expression to Alzheimer’s disease, Parkinson’s disease, and schizophrenia, with some receptors modulating microglial phagocytosis or tau phosphorylation. This study establishes, for the first time, a direct linear signaling axis connecting a brain-expressed olfactory receptor to the structural machinery of the neuron itself. It also hints at a possible bridge to tau biology, since a homolog of IGF2BP3 binds tau mRNA and IGF2BP3 variants correlate with tau burden in Alzheimer’s patients. The authors are appropriately restrained about the disease connection, emphasizing that their findings are in vitro and that the relevance to Alzheimer’s pathology remains speculative. Future work, they argue, should test Olfr544 in knockout and transgenic mouse models, identify the receptor’s endogenous brain ligands, and determine whether modulating the pathway affects synaptic integrity, neurodegeneration, or cognition. If those experiments pan out, a receptor once thought to exist solely to smell the world may turn out to be a lever for protecting the brain from one of its most devastating diseases.

Subject of Research: The role of the extra-olfactory olfactory receptor Olfr544 in neuronal neurite outgrowth and CREB signaling

Article Title: Role of olfactory receptor Olfr544 in neuronal cells: Insights into neurite outgrowth and CREB signaling

Article References: Lv, S., Liu, J., Zhou, Y., Yang, R., Zhang, T., Deng, Y., Chen, Y., Lai, C., & Chen, Q. (2026). Role of olfactory receptor Olfr544 in neuronal cells: Insights into neurite outgrowth and CREB signaling. iScience, 29(11), Article 117757. https://doi.org/10.1016/j.isci.2026.117757

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117757

Keywords: olfactory receptors, Olfr544, azelaic acid, CREB signaling, neurite outgrowth, N2a cells, cAMP, PKA, CRISPR-Cas9, Alzheimer's disease, GPCR, IGF2BP3

The traditional route to identifying a sex pheromone has always been laborious. Researchers must typically maintain live colonies of the target insect, collect and extract the chemicals emitted by females, and then sift through a complex mixture of dozens or even hundreds of volatile compounds to determine which ones actually attract males. The timing of pheromone production matters enormously, since many species release their signals only during narrow windows of the day or night, and missing that window can mean missing the signal entirely. Once candidate compounds are isolated, each must be tested for behavioural activity, often through wind-tunnel experiments or field trials, before the true pheromone can be confirmed. For rare species, invasive pests that have just appeared in a new region, or insects that are difficult to rear in captivity, this process can stall for years, leaving pest managers without the species-specific tools that pheromone-based control depends on.

The INRAE team, working within the EXPLOR’AE Programme funded through the project known as Invoria, chose to invert this logic entirely. Rather than starting with the emitter of the chemical signal, they started with the receiver: the male moth, and more specifically, the olfactory receptors housed in his antennae that are tuned to detect the female’s scent. Their study species was the lily moth, Spodoptera picta, a strikingly coloured insect whose sexual communication had never been described. The lily moth is what biologists call a non-model organism: it is not a standard laboratory species with a rich history of prior research, and this was precisely the point. If the method could work on a poorly studied insect with no established toolkit, the researchers reasoned, it could likely be extended to many other species that currently resist conventional analysis.

The first step of the reverse approach relied on genomics. By analysing the genes of the lily moth and comparing them with those of related species, the researchers narrowed down a catalogue of candidate olfactory receptors to two promising candidates. This comparative step matters because receptor genes evolve alongside the chemicals they detect; receptors in closely related species that respond to similar pheromones often share detectable similarities in their sequences. From this genetic starting material, the team turned to artificial intelligence. Machine-learning tools were used to predict the three-dimensional structures of the two candidate receptors, generating models of the protein shapes that sit in the membranes of sensory neurons and cradle odorant molecules as they arrive from the air.

With structural models in hand, the researchers applied molecular docking, a computational technique widely used in drug discovery, to predict which volatile molecules would fit and bind within the receptors’ binding pockets. Docking simulations evaluate how two molecules can interact spatially and energetically, scoring the likelihood that a given compound will make stable contact with a target protein. By screening a library of volatile compounds against the modelled receptors, the computational pipeline identified a specific molecule predicted to interact strongly with both candidate receptors: (Z,E)-9,11-tetradecadienyl acetate. In effect, the algorithm had proposed an answer to a question the insects had never been asked in the laboratory: what chemical does the female of this species use to signal her availability?

Prediction alone, however, is never sufficient in biology, and the team followed the computational phase with a rigorous sequence of experimental validation. First, the selected olfactory receptors were tested directly against the predicted volatile molecules using electrophysiological methods, which measure the electrical responses of sensory cells or expressed receptor proteins when exposed to chemical stimuli. The receptors responded, confirming computationally predicted interactions with real physiological activity. Next, the researchers combined physicochemical analysis with electrophysiology to verify two crucial facts: that female lily moths actually secrete the predicted compound as their sex pheromone, and that male moths can detect the volatile molecule in the air. These analytical steps confirmed that the AI-guided prediction matched what the insects themselves were doing.

The final and most demanding test was behavioural. A molecule may be detected by an antenna, but detection does not guarantee attraction; only a behavioural response demonstrates that the compound functions as a genuine pheromone in the life of the animal. In behavioural assays, the identified compound attracted male lily moths and elicited sexual behaviour, completing the chain of evidence from gene to receptor to molecule to mating behaviour. Taken together, the results show that an unknown pheromone of a non-model species could be identified through a pipeline that begins with sequencing, passes through AI-predicted receptor structures and molecular docking, and ends with classical electrophysiology and behavioural experiments. Each stage filtered and focused the search, dramatically reducing the number of candidate chemicals that needed laboratory testing.

The significance of this proof of concept extends well beyond a single moth species. Current, emerging and invasive crop pests are precisely the organisms for which pheromone identification is most urgently needed and most often missing, because invasive populations can spread faster than conventional chemical ecology can characterise them. Once a species’ sex pheromone is known, two well-established biocontrol strategies become available. Mating disruption involves flooding an area, such as a crop field or orchard, with synthetic pheromone so that males become unable to locate females, crashing reproduction without any toxic chemical. Alternatively, pheromone-baited traps can be used to estimate population density, monitor invasions, or suppress populations through mass trapping. Both approaches are highly species-specific, sparing beneficial insects such as pollinators and natural enemies of pests, and both are compatible with the increasingly strict regulations governing pesticide use in gardens, parks and botanical collections.

The lily moth itself illustrates why such tools matter. The species is not found in mainland France and is therefore not considered a pest of concern there, which made it an ethically convenient and scientifically demanding test case. Yet elsewhere in the world, Spodoptera picta is a significant pest, particularly in private gardens, public parks and botanical gardens, settings where the use of conventional pest-control products is often tightly restricted and where ornamental plants suffer real damage. A validated pheromone-based method could give managers in those regions a targeted, low-toxicity option, and the study’s authors note that this work opens up the possibility of using pheromone-based methods to control the moth. In this sense, the research delivers both a general technique and a concrete practical outcome for a real agricultural problem.

There is also a deeper scientific payoff. Pheromones function as chemical signatures of species, and the molecules a species uses to communicate sexually contribute to reproductive isolation, helping to keep related species distinct. As a result, identifying the compounds that make up the pheromones of moths and other insects offers new insights into the evolutionary history of these lineages, revealing how chemical communication has diverged and shaped speciation. By making pheromone identification faster and less dependent on scarce live insects, the reverse chemical ecology approach demonstrated here could populate the field with many more of these chemical signatures, allowing biologists to reconstruct communication systems across entire groups of species. What began as a pest-management problem thus ends as a window onto evolution, powered by artificial intelligence, and a demonstration that sometimes the fastest way to understand a signal is to study the ear rather than the voice.

Subject of Research: AI-guided prediction of insect olfactory receptors to identify moth sex pheromones for crop protection

Article Title: Crop protection: a new method for understanding insect sexual communication

Article References: Crop protection: a new method for understanding insect sexual communication. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: pheromones, chemical ecology, olfactory receptors, Spodoptera picta, biocontrol, mating disruption, molecular docking, artificial intelligence, INRAE, BMC Biology, invasive pests, lily moth