CSF metabolomics reveals alterations in one-carbon metabolism and phosphatidylcholine abundance in a methotrexate-treated juvenile rat model

Background:Although typically curative, treatment for pediatric acute lymphoblastic leukemia (ALL) is associated with neurotoxicity and leads to chemotherapy-related cognitive impairment (CRCI) in 40-70% of survivors, significantly impacting their quality of life. Methotrexate (MTX), a key component of ALL chemotherapy regimens, is a major contributor to CRCI. Using cerebrospinal fluid (CSF) metabolomics, our previous work showed that pediatric patients undergoing chemotherapy exhibited alterations in lipid metabolism, particularly phosphatidylcholines (PC). However, because ALL chemotherapy involves multiple agents, the specific contribution of MTX to these metabolic alterations remains unclear. Experimental procedures:Using a juvenile rat model designed to isolate MTX-specific effects within a pediatric-relevant context, we administered six intraperitoneal (0.5 mg/kg per dose) and four intrathecal (1 mg/kg per dose) MTX injections between 3 and 8 weeks of age. To characterize MTX-induced metabolic changes, we performed CSF metabolomics at the time of the first intraperitoneal injection and at the fourth. Five weeks after the last injection, we assessed whether MTX exposure impaired spatial and visual memory using Object Placement (OP) and novel Object Recognition (OR) behavioral tests, respectively, in comparison to PBS-treated controls. Results:MTX-treated rats exhibited spatial and visual memory impairments compared with controls. In accordance with our previous results and MTX's mechanism of action, determinants of one-carbon metabolism were downregulated in the CSF of MTX-treated animals over time. This includes S-adenosylmethionine (SAM, FC = 0.36, p-adj = 0.02) and methionine (FC = 3.24E-06, p-adj = 6.41E-06). In contrast, transsulfuration pathway metabolites such as cystathionine (FC = 3.98, p-adj = 0.001) and cysteine (FC = 2.81, p-adj = 0.02) were upregulated. Last, MTX treatment also induced alterations in lipid metabolism compared to controls, with a significant over-representation of 8 plasmalogens and 9 PC, consistent with our findings in the CSF of pediatric ALL patients undergoing chemotherapy. Conclusion:Although in use for more than eight decades, neither the mechanism of action nor the side effects of MTX are fully understood. Preclinical models therefore remain instrumental for defining MTX's contribution to chemotherapy-related neurotoxicity. This work, together with our recent findings in humans, demonstrates that CSF metabolomics may enable the early identification of patients at risk for CRCI through predictive biomarkers and guide future neuroprotective interventions.