Basic Science and Pathogenesis

Oxysterols, particularly 27-hydroxycholesterol (27-OHC), have been implicated in neurodegenerative diseases by disrupting neuronal and astrocytic function. The synaptic proteins SNAP25 and PSD95 are essential for synaptic integrity, while REST/PTBP1 plays a key role in neuronal plasticity. In astrocytes, RAGE activation and the downregulation of glutamate transporters GLT-1 and GLAST contribute to excitotoxicity. However, the precise molecular mechanisms underlying 27-OHC-induced neurotoxicity remain poorly understood.

Primary cortical neurons and astrocytes were exposed to physiological and pathological concentrations of 27-OHC. Western blot, qPCR, and immunocytochemistry were used to assess the expression of SNAP25, PSD95, REST, PTBP1, RAGE, GLT-1, and GLAST. Functional assays included calcium imaging and glutamate uptake measurements to evaluate synaptic and astrocytic responses.

Neuronal exposure to 27-OHC led to a significant reduction in SNAP25 and PSD95 expression, correlating with synaptic dysfunction. Additionally, REST was upregulated, while PTBP1 was downregulated, suggesting impaired neuronal plasticity. In astrocytes, 27-OHC induced RAGE upregulation and a marked decrease in GLT-1 and GLAST levels, leading to reduced glutamate uptake and increased extracellular glutamate concentrations. These findings indicate a dual mechanism whereby 27-OHC disrupts synaptic integrity in neurons while impairing astrocytic glutamate clearance, contributing to excitotoxicity.

Our study highlights the detrimental effects of 27-OHC on neuronal and astrocytic function, identifying key molecular targets involved in synaptic integrity and glutamate homeostasis. The observed dysregulation of SNAP25, PSD95, REST/PTBP1, and astrocytic RAGE, GLT-1, and GLAST underscores the role of oxysterol-induced neurotoxicity in neurodegenerative processes. Targeting these pathways may offer novel therapeutic strategies for conditions characterized by cholesterol dyshomeostasis and excitotoxicity.