A Smell Receptor in the Brain Drives Neurite Growth Through CREB Signaling

Olfactory receptors are best known for their job in the nose, where they detect the vast chemical vocabulary of the odorant world. Yet over the past three decades, researchers have discovered that these G protein-coupled receptors are not confined to the olfactory epithelium. They turn up in the heart, gut, kidney, pancreas, liver, and, strikingly, in the brain itself. What they are doing in these unexpected locations has remained one of the more tantalizing open questions in sensory biology. A new study published in the open-access journal iScience now provides one of the clearest answers yet for a receptor found in the central nervous system, showing that a mouse olfactory receptor called Olfr544 actively shapes neuronal structure by switching on a classic signaling cascade that governs neurite outgrowth.

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