Exploring microtubule dynamics in Alzheimer's disease: Longitudinal assessment using [11C]MPC-6827 PET imaging in rodent models of Alzheimer's-related pathology.

INTRODUCTION: Microtubule (MT) stability is crucial for proper neuronal function. Understanding MT dysregulation is critical for connecting amyloid beta (Aß) and tau-based degenerative events and early changes in presymptomatic Alzheimer's disease (AD). Herein we present positron emission tomography (PET) imaging properties of our MT-PET radiotracer, [11C]MPC-6827, in multiple established AD mouse models. METHODS: Longitudinal PET, biodistribution, autoradiography, immunohistochemistry, and behavioral studies were conducted at multiple time points in APPswe/PSEN1dE9 (APP/PS1), P301S-PS19 (P301S), 5xFAD, and age-matched control mice. RESULTS: Longitudinal [11C]MPC-6827 brain imaging showed significant increases in APP/PS1, P301S, and 5xFAD mice compared to controls. Longitudinal MT-PET correlated positively with biodistribution, autoradiography, and immunohistochemistry results and negatively with behavior data. DISCUSSION: Our study demonstrated significant longitudinal [11C]MPC-6827 PET increases in multiple AD mouse models for the first time. Strong correlations between PET and biomarker data underscored the interplay of MT destabilization, amyloid, and tau pathology in AD. These results suggest [11C]MPC-6827 PET as a promising tool for monitoring MT dysregulation early in AD progression. HIGHLIGHTS: Longitudinal positron emission tomography (PET) imaging studies using [11C]MPC-6827 in multiple established Alzheimer's disease (AD) mouse models revealed an early onset of microtubule dysregulation, with significant changes in brain radiotracer uptake evident from 2 to 4 months of age. Intra-group analysis showed a progressive increase in microtubule dysregulation with increasing AD burden, supported by significant correlations between PET imaging data and biodistribution, autoradiography, and molecular pathological markers. [11C]MPC-6827 PET imaging demonstrated its efficacy in detecting early microtubule alterations preceding observable behavioral changes in AD mouse models, suggesting its potential for early AD imaging. The inclusion of the 5xFAD mouse model further elucidated the impact of amyloid beta (Aß) toxicity on inducing tau hyperphosphorylation-mediated microtubule dysregulation, highlighting the versatility of [11C]MPC-6827 in delineating various aspects of AD pathology. Our study provides immediate clarity on high uptake of the microtubule-based radiotracer in AD brains in a longitudinal setting, which directly informs clinical utility in Aß/tau-based studies.

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

The microtubule (MT) instability observed in Alzheimer's disease (AD) is commonly attributed to hyperphosphorylation of the MT-associated protein, tau. In vivo PET imaging offers an opportunity to gain critical information about MT changes with the onset and development of AD and related dementia. We developed the first brain-penetrant MT PET ligand, [11C]MPC-6827, and evaluated its in vivo imaging utility in vervet monkeys. Consistent with our previous in vitro cell uptake and in vivo rodent imaging experiments, [11C]MPC-6827 uptake increased with MT destabilization. Radioactive uptake was inversely related to (cerebrospinal fluid) CSF Aß42 levels and directly related to age in a nonhuman primate (NHP) model of AD. Additionally, in vitro autoradiography studies also corroborated PET imaging results. Here, we report the preliminary results of PET imaging with [11C]MPC-6827 in four female vervet monkeys with high or low CSF Aß42 levels, which have been shown to correlate with the Aß plaque burden, similar to humans.

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

G-protein-coupled receptor 119 (GPR119) has emerged as a promising target for treating type 2 diabetes mellitus. Activating GPR119 improves glucose homeostasis, while suppressing appetite and weight gain. Measuring GPR119 levels in vivo could significantly advance GPR119-based drug development strategies including target engagement, occupancy, and distribution studies. To date, no positron emission tomography (PET) ligands are available to image GPR119. In this paper, we report the synthesis, radiolabeling, and preliminary biological evaluations of a novel PET radiotracer [18F]KSS3 to image GPR119. PET imaging will provide information on GPR119 changes with diabetic glycemic loads and the efficacy of GPR119 agonists as antidiabetic drugs. Our results demonstrate [18F]KSS3's high radiochemical purity, specific activity, cellular uptake, and in vivo and ex vivo uptake in pancreas, liver, and gut regions, with high GPR119 expression. Cell pretreatment with nonradioactive KSS3, rodent PET imaging, biodistribution, and autoradiography studies showed significant blocking in the pancreas showing [18F]KSS3's high specificity.

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

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.

[18F]KS1, a novel ascorbate-based ligand images ROS in tumor models of rodents and nonhuman primates.

Over production of reactive oxygen species (ROS) caused by altered redox regulation of signaling pathways is common in many types of cancers. While PET imaging is recognized as the standard tool for cancer imaging, there are no clinically-approved PET radiotracers for ROS-imaging in cancer diagnosis and treatment. An ascorbate-based radio ligand promises to meet this urgent need. Our laboratory recently synthesized [18F] KS1, a fluoroethoxy furanose ring-containing ascorbate derivative, to track ROS in prostate tumor-bearing mice. Here we report cell uptake assays of [18F]KS1 with different ROS-regulating agents, PET imaging in head and neck squamous cell carcinoma (HNSCC) mice, and doxorubicin-induced rats; PET imaging in healthy and irradiated hepatic tumor-bearing rhesus to demonstrate its translational potential. Our preliminary evaluations demonstrated that KS1 do not generate ROS in tumor cells at tracer-level concentrations and tumor-killing properties at pharmacologic doses. [18F]KS1 uptake was low in HNSCC pretreated with ROS blockers, and high with ROS inducers. Tumors in high ROS-expressing SCC-61 took up significantly more [18F]KS1 than rSCC-61 (low-ROS expressing HNSCC); high uptake in doxorubicin-treated rats compared to saline-treated controls. Rodent biodistribution and PET imaging of [18F]KS1 in healthy rhesus monkeys demonstrated its favorable safety, pharmacokinetic properties with excellent washout profile, within 3.0 h of radiotracer administration. High uptake of [18F]KS1 in liver tumor tissues of the irradiated hepatic tumor-bearing monkey showed target selectivity. Our strong data in vitro, in vivo, and ex vivo here supports the high translational utility of [18F]KS1 to image ROS.

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

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, ß, 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 ß 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.

Relapse-like behavior and nAChR sensitization following intermittent access nicotine self-administration.

Many tobacco smokers consume nicotine intermittently, but the underlying mechanisms and neurobiological changes associated with intermittent nicotine intake are unclear. Understanding intermittent nicotine intake is a high priority, as it could promote therapeutic strategies to attenuate tobacco consumption. We examined nicotine intake behavior and neurobiological changes in male rats that were trained to self-administer nicotine during brief (5 min) trials interspersed with longer (15 min) drug-free periods. Rats readily adapted to intermittent access (IntA) SA following acquisition on a continuous access (ContA) schedule. Probabilistic analysis of IntA nicotine SA suggested reduced nicotine loading behavior compared to ContA, and nicotine pharmacokinetic modeling revealed that rats taking nicotine intermittently may have increased intake to maintain blood levels of nicotine that are comparable to ContA SA. After IntA nicotine SA, rats exhibited an increase in unreinforced responses for nicotine-associated cues (incubation of craving) and specific alterations in the striatal proteome after 7 days without nicotine. IntA nicotine SA also induced nAChR functional upregulation in the interpeduncular nucleus (IPN), and it enhanced nicotine binding in the brain as determined via [11C]nicotine positron emission tomography. Reducing the saliency of the cue conditions during the 5 min access periods attenuated nicotine intake, but incubation of craving was preserved. Together, these results indicate that IntA conditions promote nicotine SA and nicotine seeking after a nicotine-free period.

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

Purpose: Microtubules (MTs) are structural units made of α and ß 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). Methods: Cell 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. Results: Cocaine-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. Conclusions: This 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.