Preliminary mechanistic insights of a brain-penetrant microtubule imaging PET ligand in a tau-knockout mouse model

Abstract

BackgroundMicrotubules (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.MethodsIn 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.ResultsCell 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.ConclusionsCollectively, 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.