Dynamic Proteomic and Metabolomic Analysis Reveals Metabolic Reprogramming During Early Neuronal Transdifferentiation of Human Fibroblasts Driven by Forskolin

Abstract

Forskolin (FSK) is a well-characterized small-molecule activator of adenylyl cyclase that drives direct neuronal transdifferentiation in human fibroblasts; however, the temporal sequence and coordinated relationships among proteomic and metabolic adaptations during the initiation phase of lineage conversion remain poorly understood. In this study, we applied data-independent acquisition (DIA)-based quantitative proteomics and untargeted metabolomics on BJ human dermal fibroblasts at three biological timepoints: pre-induction (day 0), commitment onset (day 2), and neuronal maturation (day 5). Under the established FSK-based induction protocol, BJ fibroblasts rapidly acquired neuronal-like features, with more than 90% of cells becoming TUJ1-positive by day 5. Proteomic profiling revealed a profound, dichotomous regulatory shift: time-dependent activation of core metabolic and energy pathways (glycolysis, the TCA cycle, and oxidative phosphorylation) coupled with persistent suppression of cell-cycle progression and DNA replication. Concordantly, global metabolomic profiling revealed a statistically unidirectional transition in metabolic states characterized by the progressive accumulation of phosphoenolpyruvate (PEP). Collectively, our findings identify coordinated remodeling of central carbon metabolism as a prominent early molecular feature associated with neuronal transdifferentiation under the FSK-based induction protocol. This study provides an integrated proteomic and metabolomic framework for understanding early molecular remodeling during chemically induced cell fate conversion and provides a basis for future functional studies of metabolic regulation during reprogramming.