PET imaging of alpha-synuclein: from radiotracer design through in vitro and in vivo translation

The development of positron emission tomography (PET) tracers targeting α-synuclein (α-syn) aggregates remains a major challenge in PET imaging of neurodegenerative diseases. This review provides a comprehensive overview of the recent advances, key obstacles, and aims to give future directions for the development of α-syn PET tracers. The first part of the review focuses on the experimental strategies to develop potential α-syn PET ligands. We overview the differences between various types of α-syn fibrils, including preformed fibrils and patient-derived fibrils, and methods such as solid-state nuclear magnetic resonance and cryogenic electron microscopy used for structure elucidation of the fibrils. Furthermore, the review summarizes the techniques for the assessment of ligand binding to α-syn, such as fibril binding assays (competition and saturation binding assays), macro- and microautoradiography, and alternative methods like surface plasmon resonance and biolayer interferometry. Determination of pharmacokinetics and metabolism are likewise important steps in α-syn tracer development, and hurdles and merits of in vitro and in vivo methods are contemplated, in the context of translation to in vivo evaluation in fibril-inoculated and transgenic animal models. Finally, off-target binding of tracer candidates is described, which still remains one of the major pitfalls of α-syn-targeting PET tracers. The second part of the review overviews all small molecule α-syn PET tracers developed since 2022, highlighting their progress, current limitations, and future directions for achieving clinically viable α-syn PET imaging agents.