Evidence of Citicoline Cytoprotection Through Intracellular Proteome Remodeling Processes, Proteostatic Balance and Hormesis by an Unbiased Shot-Gun Proteomic Study

Abstract

Citicoline, also known as CDP-choline is a nootropic agent used for the cure of glaucoma and currently tested as a first-line treatment through a multi-center, international, phase III, randomized clinical trial with citicoline eyedrops (see ClinicalTrials.gov: ID NCT05710198).Several clinical and preclinical studies have associated the neuroenhancement and neuroprotective effects of citicoline to its role as metabolic precursor of structural and functional components of the cell membrane, such as phosphatidylcholine and sphingomyelin, and of neurotransmitters. However, there is compelling evidence that the molecular mechanisms underscoring its cytoprotective activity include additional pharmacological activities uncharacterized yet.To shed further light on its pharmacology, we here sought to investigate the effect of delivering two doses of citicoline (0.1 µM and 1 mM), previously associated with cyto-protection, on the global proteome of neuroblastoma cells by an unbiased shot-gun proteomics approach.With >4000 unique proteins identified and quantified per experimental condition, the proteomics approach has retrieved evidence that citicoline induces a robust remodeling of the intracellular proteome. Data clustering and rationalization highlighted that citicoline stimulated a robust enrichment of main protein clusters, including mRNA spicing, mitochondrial metabolism, components of the Ubiquitin Proteasome System (UPS) and additional factors involved in the proteostasis network balance, cyto-protection and signaling pathways.These proteomics findings introduce 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 support the rationale of clinical usage in neurodegenerative processes and human disorders characterized by proteotoxicity.

Note:
Funding declaration: The authors acknowledge the Ministry Health an Fondazione Roma for their support. Furthermore, the authors acknowledge Omikron Italia srl for its support to the study. The authors acknowledge MUR Prin 2022 n. 2022R9WCZS_001.

Conflict of Interests: The authors declare that they have no known competing financial interests or personal relationships thatcould have appeared to influence the work reported in this paper.

Keywords: Citicoline, glaucoma, Proteomics, Proteostasis, Proteasome