Proteomic Profiling of Primary Hippocampal Neurons Reveals Noncanonical GFAP Expression and Metabolic Adaptations in Glia-Free Culture.

Despite their widespread use as a research model, a comprehensive, quantitative proteomic profile of the cultured hippocampal neurons has remained unexplored. Here, we provide the first global proteomic characterization of primary murine hippocampal neurons cultured for 14 days under near-physiological glucose conditions (2.5 mM). Using label-free mass spectrometry and the Total Protein Approach (TPA), we quantified over 5,500 proteins, revealing a metabolic signature consistent with oxidative phosphorylation and a neuron-specific proteome largely devoid of astrocytic markers. Unexpectedly, glial fibrillary acidic protein (GFAP) was highly abundant in pure neuronal cultures, and its knockdown impaired neuronal viability and morphology. These findings suggest a compensatory role for GFAP in neurons cultured without astrocytic support. Additionally, our data highlight significant underexpression of key synaptic proteins compared to hippocampal tissue, underscoring the limitations of neuron-only culture systems. This study establishes a new molecular reference for hippocampal neurons and offers insights into neuronal adaptation in simplified environments.