Citicoline Triggers Proteome Remodeling and Proteostatic Adaptation: Evidence from Shotgun Proteomics

Background/Objectives: Citicoline, also known as CDP-choline, is a nootropic agent currently used in the treatment of glaucoma and is undergoing evaluation as a first-line therapy in a multi-center, international, phase III, randomized clinical trial involving citicoline eyedrops (ClinicalTrials.gov ID: NCT05710198). Numerous clinical and preclinical studies have linked the neuroenhancement and neuroprotective effects of citicoline to its role as a metabolic precursor for structural and functional components of cell membranes (such as phosphatidylcholine and sphingomyelin) and for neurotransmitters (e.g., acetylcholine and dopamine). However, compelling evidence suggests that the molecular mechanisms underlying its cytoprotective activity involve additional as-yet uncharacterized pharmacological actions. Methods: To further elucidate its pharmacology, we investigated the effect of two cytoprotective doses of citicoline (0.1 mM and 1 mM) on the global proteome of neuroblastoma cells using an unbiased shotgun proteomics approach. Results: With over 4000 unique proteins identified and quantified per experimental condition, the proteomics analysis revealed that citicoline, after 6 h of stimulation, induces a profound and robust remodeling of the intracellular proteome compared to untreated cells. Importantly, this effect was observed to significantly diminish by 18 h of stimulation, highlighting its transient nature (data are available via ProteomeXchange with identifier PXD061053). The clustering and rationalization of proteins upregulated by citicoline treatment identified the enrichment of key pathways for mRNA splicing, protein translation, proteostasis balance through the ubiquitin proteasome system (UPS), and mitochondrial metabolism. Conclusions: These proteomics findings introduce previously uncharacterized biological effects of citicoline and foster the working hypothesis that this drug may exert its cytoprotective activity through molecular mechanisms linked to the hormesis principle. These data further support the rationale for its clinical application in neurodegenerative processes and human disorders characterized by proteotoxicity.