Disruption of dopamine transmission by cholesterol depletion is associated with alterations in protein lipid raft partitioning and actin dynamics

Disrupted brain cholesterol homeostasis is implicated in neurological disorders involving aberrant dopamine (DA) signaling; however, the direct effects of cholesterol on DA transmission in native tissue have not yet been demonstrated. Using ex vivo fast-scan cyclic voltammetry in nucleus accumbens slices from male rats, we found that membrane cholesterol depletion with methyl-β-cyclodextrin (MβCD, 3-10 mM) significantly reduced evoked DA release and decreased the apparent maximal rate of DA reuptake via the dopamine transporter (DAT). Because cholesterol is critical for the formation of lipid raft microdomains, cholesterol depletion could disrupt DA transmission by altering the membrane localization of proteins involved in neurotransmitter release. Using sucrose density gradient fractionation, we found that MβCD decreased the raft association of vesicle-associated membrane protein 2 (VAMP2) without altering the localization of syntaxin-1A, synaptosomal-associated protein 25, synaptotagmin-1, and N-type voltage-gated calcium channels. Therefore, MβCD may reduce DA release by disrupting localization of VAMP2, a core component of the vesicle fusion machinery. We also examined actin polymerization, a key regulator of vesicle docking and fusion, and found that MβCD treatment decreased actin polymerization, as evidenced by an increased globular-to-filamentous actin ratio and reduced phalloidin labeling of filamentous actin in striatal slices. Finally, although DAT lipid raft localization was unchanged, MβCD attenuated cocaine's ability to inhibit DAT reuptake function, suggesting that cholesterol depletion disrupts the outward-facing conformation of DAT required for high-affinity ligand binding. Overall, these findings provide new mechanistic insights into how cholesterol depletion may contribute to dysregulated DA signaling in diseases involving altered brain cholesterol metabolism.