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

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.

Preliminary PET Imaging of Microtubule-Based PET Radioligand [11C]MPC-6827 in a Nonhuman Primate Model of Alzheimer’s Disease

“…Our prior mechanistic studies in tau knockout mouse models showed that [ 11 C]­MPC-6827 is more selective for destabilized MTs, and this preliminary investigation in four vervets with different CSF Aβ values also supports this interpretation. High-radiotracer uptake was observed in regions commonly associated with high tau and amyloid plaque densities. , Current preliminary results encourage the study PET imaging of the [ 11 C]­MPC-6827 radiotracer in more subjects of the AD model of NHPs to validate the results statistically and to establish the MT destabilization model for the AD pathogenesis.…”

“…Our prior mechanistic studies in tau knockout mouse models 51 showed that [ 11 C]MPC-6827 is more selective for destabilized MTs, and this preliminary investigation in four…”

Binding Parameters of [11C]MPC-6827, a Microtubule-Imaging PET Radiopharmaceutical in Rodents

“…Additionally, our new in vitro, in vivo and ex vivo mechanistic studies showed that [ 11 C]MPC-6827 uptake is higher to destabilized MTs than stabilized MTs and that the uptake increases with brain Aβ/tau burden [31]. Postmortem studies on patients with confirmed AD showed higher radiopharmaceutical uptake than the cognitively normal age-matched controls [31]. Given the extensive work published from our group on [ 11 C]MPC-6827, the next steps would be to quantify its binding and metabolic characteristics in target (brain) and plasma-these data will strengthen its potential as a CNS PET radiopharmaceutical.…”

“…To study MT destabilization and its progression in all dementia disorders including AD, we developed the first brain-penetrating PET radiopharmaceutical, [ 11 C]MPC-6827 [27], and evaluated its imaging efficacy in rodents and nonhuman primate models of AD [27][28][29][30]. Additionally, our new in vitro, in vivo and ex vivo mechanistic studies showed that [ 11 C]MPC-6827 uptake is higher to destabilized MTs than stabilized MTs and that the uptake increases with brain Aβ/tau burden [31]. Postmortem studies on patients with confirmed AD showed higher radiopharmaceutical uptake than the cognitively normal age-matched controls [31].…”

First GPR119 PET Imaging Ligand: Synthesis, Radiochemistry, and Preliminary Evaluations

“…To demonstrate that our synthesized [ 18 F]KSS3 can be used as in vivo PET imaging agent, we performed small animal microPET imaging using TriFoil microPET/CT system. 50 Male C57BL/J mice (n = 4, 22 ± 3 g) were anesthetized in an induction chamber with 2−3% isoflurane in oxygen, and a catheter was placed in the lateral tail vein. Each mouse was secured to a custom-designed acrylic bed equipped with a nosecone for gas anesthesia.…”