PROJECT SUMMARY Around 50 million people worldwide are suffering from Alzheimer’s disease (AD) and related dementias, and AD is observed primarily in aged people. Current diagnosis of AD relies primarily on observations of declines in mental and cognitive functions, when irreversible brain damage occurs. Positron emission tomography (PET) tracers for early diagnosis of AD have been developed via assessing Aβ or tau levels, however aggregate-targeting tracers often suffer from high nonspecific binding. Therefore, there is an urgent need for developing novel tools targeting molecular level to improve the specificity of early diagnosis of AD. Cellular senescence has been shown to be a critical contributor disrupting the homeostasis and functions of aging brains. In aged brains, senescent cells accumulate and exert chronic inflammatory pressure on surrounding cells. Higher levels of senescence in different types of brain cells have been observed in mice and human with AD and selective removal of senescent cells reduces Aβ plaque formation in mouse brain and improves cognitive functions. These findings indicate that senescence can potentially serve as an early indicator and therapeutic target for AD. However, tools for real-time in vivo senescence detection are extremely limited. Recently, the Cui group at the University of Florida (UF) has developed a series of activatable molecular probes, enabling the successful real-time imaging of senescence in animal models for the first time. These probes have high detection sensitivity produced by a self-immobilizing moiety installed on the probe so that upon activation, the probe can be covalently linked to surrounding proteins at the site of activation. Based on this strategy, SenoTrac, partnering with UF and Moffitt Cancer Center, aims to develop the first- in-class PET probes for senescence. Briefly, the novel PET probe will consist of a β-galactoside that can be specifically cleaved by senescence-associated β-galactosidase (SA-β-gal), a 18F, and a self-immobilizing moiety that can anchor the probe onto surrounding proteins upon activation. We expect the activatable PET probe to have high brain uptake, minimal diffusion and high signal/background contrast with excellent spatial resolution. We will characterize these probes in vitro (SenoTrac and UF), and we will also evaluate their ability to cross the blood brain barrier in animals (UF). Radiochemistry before PET imaging will be carried at Moffitt. AD mouse model (5xFAD) will be used in the dynamic PET/CT scan to see the levels of senescence in different age groups. Additionally, we will clear senescent cells using senolytics, perform PET imaging of senescence and evaluate AD phenotypes in these mice. Ex vivo analysis of the brain tissues will provide information of other AD and senescence biomarkers (UF). We expect to draw correlation of senescence levels and various AD phenotypes. In Phase 1, we will develop the PET probes for senescence tracking in AD animal models. In Phase 2, we will evaluate the probes in different animal models, and a small-scale human trial. We will navigate the regulatory requirements, and work towards commercialization of these probes.

PROJECT NARRATIVE Public Health Relevance Statement: Millions of people worldwide suffer from incurable Alzheimer’s disease, over 95% of which is seen in people aged 65 or older, and the number continues to grow as the population ages. Senescence, a cellular state with cell cycle arrest, is tightly linked to Alzheimer’s disease, and real-time detection of senescence can potentially be used as an early diagnosis for Alzheimer’s disease. The proposed work will develop and evaluate positron emission tomography (PET) imaging probes for senescence detection in the brain using animal models, and eventually clinically translate them for human use in the future.