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Abstract Background Microtubules (MTs) are critical for cell structure, function, and survival. MT instability may contribute to Alzheimer’s disease (AD) pathogenesis as evidenced by persistent negative regulation (phosphorylation) of the neuronal microtubule-associated protein tau. Hyperphosphorylated tau, not bound to MTs, forms intraneuronal pathology that correlates with dementia and can be tracked using positron emission tomography (PET) imaging. The contribution of MT instability in AD remains unknown, though it may be more proximal to neuronal dysfunction than tau accumulation. Our lab reported the first brain-penetrant MT-based PET ligand, [11C]MPC-6827, and its PET imaging with this ligand in normal rodents and non-human primates demonstrated high brain uptake and excellent pharmacokinetics. Target engagement and mechanism of action using in vitro, in vivo, and ex vivo methods were evaluated here. Methods In vitro cell uptake assay was performed in SH-SY5Y neuronal cells with [11C]MPC-6827, with various MT stabilizing and destabilizing agents. To validate the in vitro results, wild type (WT) mice (n = 4) treated with a brain-penetrant MT stabilizing drug (EpoD) underwent microPET/CT brain imaging with [11C]MPC-6827. To determine the influence of tau protein on radiotracer binding in the absence of protein accumulation, we utilized tau knockout (KO) mice. In vivo microPET imaging, ex vivo biodistribution, and autoradiography studies were performed in tau KO and WT mice (n = 6/group) with [11C]MPC-6827. Additionally, α, β, and acetylated tubulin levels in both brain samples were determined using commercially available cytoskeleton-based MT kit and capillary electrophoresis immunoblotting assays. Results Cell uptake demonstrated higher radioactive uptake with MT destabilizing agents and lower uptake with stabilizing agents compared to untreated cells. Similarly, acute treatment with EpoD in WT mice decreased [11C]MPC-6827 brain uptake, assessed with microPET/CT imaging. Compared to WT mice, tau KO mice expressed significantly lower β tubulin, which contains the MPC-6827 binding domain, and modestly lower levels of acetylated α tubulin, indicative of unstable MTs. In vivo imaging revealed significantly higher [11C]MPC-6827 uptake in tau KOs than WT, particularly in AD-relevant brain regions known to express high levels of tau. Ex vivo post-PET biodistribution and autoradiography confirmed the in vivo results. Conclusions Collectively, our data indicate that [11C]MPC-6827 uptake inversely correlates with MT stability and may better reflect the absence of tau than total tubulin levels. Given the radiotracer binding does not require the presence of aggregated tau, we hypothesize that [11C]MPC-6827 may be particularly useful in preclinical stages of AD prior to tau deposition. Our study provides immediate clarity on high uptake of the MT-based radiotracer in AD brains, which directly informs clinical utility in MT/tau-based PET imaging studies.

PurposeMicrotubules (MTs) are structural units made of α and β tubulin subunits in the cytoskeleton responsible for axonal transport, information processing, and signaling mechanisms—critical for healthy brain function. Chronic cocaine exposure affects the function, organization, and stability of MTs in the brain, thereby impairing overall neurochemical and cognitive processes. At present, we have no reliable, non-invasive methods to image MTs for cocaine use disorder (CUD). Recently we reported the effect of cocaine in patient-derived neuroblastoma SH-SY5Y cells. Here we report preliminary results of a potential imaging biomarker of CUD using the brain penetrant MT-based radiotracer, [11C]MPC-6827, in an established rodent model of cocaine self-administration (SA).MethodsCell uptake studies were performed with [11C]MPC-6827 in SH-SY5Y cells, treated with or without cocaine (n = 6/group) at 30 and 60 min incubations. MicroPET/CT brain scans were performed in rats at baseline and 35 days after cocaine self-administration and compared with saline-treated rats as controls (n = 4/sex). Whole-body post-PET biodistribution, plasma metabolite assay, and brain autoradiography were performed in the same rats from imaging.ResultsCocaine-treated SH-SY5Y cells demonstrated a ∼26(±4)% decrease in radioactive uptake compared to non-treated controls. Both microPET/CT imaging and biodistribution results showed lower (∼35 ± 3%) [11C]MPC-6827 brain uptake in rats that had a history of cocaine self-administration compared to the saline-treated controls. Plasma metabolite assays demonstrate the stability (≥95%) of the radiotracer in both groups. In vitro autoradiography also demonstrated lower radioactive uptake in cocaine rats compared to the control rats. [11C]MPC-6827’s in vitro SH-SY5Y neuronal cell uptake, in vivo positron emission tomography (PET) imaging, ex vivo biodistribution, and in vitro autoradiography results corroborated well with each other, demonstrating decreased radioactive brain uptake in cocaine self-administered rats versus controls. There were no significant differences either in cocaine intake or in [11C]MPC-6827 uptake between the male and female rats.ConclusionsThis project is the first to validate in vivo imaging of the MT-associations with CUD in a rodent model. Our initial observations suggest that [11C]MPC-6827 uptake decreases in cocaine self-administered rats and that it may selectively bind to destabilized tubulin units in the brain. Further longitudinal studies correlating cocaine intake with [11C]MPC-6827 PET brain measures could potentially establish the MT scaffold as an imaging biomarker for CUD, providing researchers and clinicians with a sensitive tool to better understand the biological underpinnings of CUD and tailor new treatments.

Impairment and/or destabilization of neuronal microtubules (MTs) resulting from hyper-phosphorylation of the tau proteins is implicated in many pathologies, including Alzheimer’s disease (AD), Parkinson’s disease and other neurological disorders. Increasing scientific evidence indicates that MT-stabilizing agents protect against the deleterious effects of neurodegeneration in treating AD. To quantify these protective benefits, we developed the first brain-penetrant PET radiopharmaceutical, [11C]MPC-6827, for in vivo quantification of MTs in rodent and nonhuman primate models of AD. Mechanistic insights revealed from recently reported studies confirm the radiopharmaceutical’s high selectivity for destabilized MTs. To further translate it to clinical settings, its metabolic stability and pharmacokinetic parameters must be determined. Here, we report in vivo plasma and brain metabolism studies establishing the radiopharmaceutical-binding constants of [11C]MPC-6827. Binding constants were extrapolated from autoradiography experiments; pretreatment with a nonradioactive MPC-6827 decreased the brain uptake >70%. It exhibited ideal binding characteristics (typical of a CNS radiopharmaceutical) including LogP (2.9), Kd (15.59 nM), and Bmax (11.86 fmol/mg). Most important, [11C]MPC-6827 showed high serum and metabolic stability (>95%) in rat plasma and brain samples.