Fluoxetine promotes lysosome biogenesis via regulation of a RIOK2-TFEB axis

• Discovery of two novel TFEB phosphorylation sites (S74 and S151) important for TFEB translocation and activation. • Uncovering the RIOK2–TFEB signaling axis as a new pathway controlling lysosome biogenesis. • Demonstration that fluoxetine enhances lysosome-dependent clearance of lipid droplets and toxic protein aggregates. Lysosomes are central to cellular proteostasis and metabolic homeostasis, and their dysfunction contributes to neurodegenerative and metabolic disorders. The transcription factor TFEB coordinates lysosomal biogenesis and autophagy through phosphorylation-dependent regulation. Based on our previous finding that the dopamine transporter inhibitor LH2-051 activates TFEB and enhances lysosomal biogenesis, we asked whether other neurotransmitter transporter inhibitors exert similar effects. Here, we identify fluoxetine, a selective serotonin reuptake inhibitor (SSRI), as a new modulator of the TFEB–lysosome axis. Fluoxetine promotes TFEB nuclear translocation and lysosomal gene expression independently of mTORC1, GSK3β, CDK9, or serotonergic signaling. Mechanistically, fluoxetine induces dephosphorylation of TFEB at Ser74 and Ser151 by inhibiting the atypical kinase RIOK2, revealing a previously unrecognized RIOK2–TFEB regulatory pathway. Fluoxetine-induced activation of TFEB enhances lysosome-dependent degradation of lipid droplets and pathogenic protein aggregates (Aβ, α-synuclein, and polyQ). These findings uncover a serotonin-independent biological action of fluoxetine and suggest that neurotransmitter transporter inhibitors may constitute a pharmacological class that can systemically enhance lysosomal function in neurodegenerative disease.