Effect of ethanol and cocaine on [11C]MPC-6827 uptake in SH-SY5Y cells

Abstract

Microtubules (MTs) are structural units in the cytoskeleton. In brain cells they are responsible for axonal transport, information processing, and signaling mechanisms. Proper function of these processes is critical for healthy brain functions. Alcohol and substance use disorders (AUD/SUDs) affects the function and organization of MTs in the brain, making them a potential neuroimaging marker to study the resulting impairment of overall neurobehavioral and cognitive processes. Our lab reported the first brain-penetrant MT-tracking Positron Emission Tomography (PET) ligand [11C]MPC-6827 and demonstrated its in vivo utility in rodents and non-human primates. To further explore the in vivo imaging potential of [11C]MPC-6827, we need to investigate its mechanism of action. Here, we report preliminary in vitro binding results in SH-SY5Y neuroblastoma cells exposed to ethanol (EtOH) or cocaine in combination with multiple agents that alter MT stability. EtOH and cocaine treatments increased MT stability and decreased free tubulin monomers. Our initial cell-binding assay demonstrated that [11C]MPC-6827 may have high affinity to free/unbound tubulin units. Consistent with this mechanism of action, we observed lower [11C]MPC-6827 uptake in SH-SY5Y cells after EtOH and cocaine treatments (e.g., fewer free tubulin units). We are currently performing in vivo PET imaging and ex vivo biodistribution studies in rodent and nonhuman primate models of AUD and SUDs and Alzheimer's disease.

Initial Evaluations of the Microtubule-Based PET Radiotracer, [11C]MPC-6827 in a Rodent Model of Cocaine Abuse

“…These assays are used to demonstrate a proof-of-principle specificity, which often do not show complete blocking. MPC-6827 primarily targets the β tubulin sites at pharmacological doses ( 14 , 25 , 26 ). The lowered radioactive uptake in cocaine-treated SH-SY5Y cells indicates that [ 11 C]MPC-6827 may be tracking the destabilized free β tubulin units, as cocaine treatment decreased free tubulin content in the same cells.…”

“…Cells maintain a balance between stabilized polymerized MTs and destabilized α/β free tubulin units in the cytoplasm ( 33 ). We recently reported [ 11 C]MPC-6827’s in vitro selectivity toward destabilized tubulin units using various MT agents including stabilizing and destabilizing agents in patient-derived SH-SY5Y neuroblastoma cells ( 14 ). Using MT-based assay (Cytoskeleton Inc, Denver, CO) cocaine-treated cells showed ∼21% less destabilized MT content than untreated cells, indicating that cocaine exposure compromises cellular MT stability, ( 34 – 36 ) treated with cocaine and untreated cells ( 14 ).…”

“…We recently reported [ 11 C]MPC-6827’s in vitro selectivity toward destabilized tubulin units using various MT agents including stabilizing and destabilizing agents in patient-derived SH-SY5Y neuroblastoma cells ( 14 ). Using MT-based assay (Cytoskeleton Inc, Denver, CO) cocaine-treated cells showed ∼21% less destabilized MT content than untreated cells, indicating that cocaine exposure compromises cellular MT stability, ( 34 – 36 ) treated with cocaine and untreated cells ( 14 ). After demonstrating changes in MT integrity in vitro with cocaine, our next step was to determine whether [ 11 C]MPC-6827 could detect the same MT alterations in the same cells.…”

“…We recently demonstrated that cocaine treatment in patient-derived SH-SY5Y neuroblastoma cells changes the MT integrity ( 14 ). Cocaine treated cells showed ∼24% fewer free/unbound tubulin units than the untreated cells.…”

“…Recently, we reported the automated radiochemical synthesis of [ 11 C]MPC-6827 as the first brain-penetrating MT PET ligand and demonstrated its in vivo PET imaging utility in cocaine-naïve rodents and non-human primates ( 12 , 13 ). Cell uptake results in a patient-derived neuroblastoma SH-SY5Y cells with cocaine treatment showed that [ 11 C]MPC-6827 binds selectively to destabilized tubulin units ( 14 ). Therefore, direct in vivo imaging of MT levels could reveal real-time cytoskeletal changes during CUD progression.…”