Final Report Summary - PRISTINE-PD (Prion-like transmission of α-synuclein in Parkinson's disease) Around 10 years ago, Braak and coworkers described that in Parkinson’s disease (PD) intracellular aggregates (called Lewy bodies and neurites) of the protein alpha-synuclein (a-syn) first appear in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve. This occurs several years prior to Lewy pathology appearing in the substantia nigra dopaminergic neurons, which are the neurons crucial for the classical motor symptoms of PD. The idea of an unidentified "neurotropic virus", propagating Lewy pathology between brain regions in PD, was discussed. In 2008, we (and others) observed Lewy pathology in grafted embryonic neurons in the brains of patients who had undergone surgery more than a decade before death. This put the idea of a prion-like mechanism in PD, involving misfolded a-syn, at the center stage of research. The PRISTINE-PD project explores the hypothesis that a-syn acts in a prion-like fashion and thereby underpins the slow and progressive spreading of neuropathology between brain regions in PD.During two and a half years (the project was terminated prematurely due to the principal investigator moving outside Europe), we conducted 8 interconnected subprojects aimed at understanding how a-syn can transfer between nerve cells and what the consequences are. We have found that the interactions between a-syn and lipids are probably important for the aggregation of a-syn. Some a-syn appears to be packaged into small vesicles called exosomes in conjunction with the transfer process. We have generated several cell culture models that allow us to monitor cell-to-cell a-syn transfer in a quantitative fashion using automated technology. We have been able to pinpoint that a process called endocytosis is important in the cellular uptake of a-syn. At the end of the funding period, we were poised to use the cell culture models to screen for molecules that inhibit a-syn uptake, and potentially could be developed into drugs that slow the progression of PD. We have developed four new paradigms in mice that allow us to study a-syn transfer in vivo. First, we injected different molecular forms of a-syn into the olfactory system of mice. Here we can follow the time course of anatomical spread in brain regions affected by PD, as well as if and when a-syn aggregates are formed. We were also able to detect olfactory deficits following the injection of certain a-syn forms. Second, we promoted inflammation in the gut of mice in order to cause misfolding of a-syn in gut nerves, with the idea that the misfolded protein will eventually transfer to the brain. Third, we used intracerebral grafts of nerve cells into mice overexpressing a-syn and followed the uptake of a-syn in the grafted neurons. In this model we used genetically modified donor tissue and at the end of the funding period we were ready to pinpoint what molecular pathways influence a-syn transfer. Fourth, we transplanted brain tissue into the anterior chamber of the eye of mice. Using two-photon confocal microscopy, we could visualize these grafted cells for several months and subject them to injections of different forms of a-syn in the anterior chamber.We have also generated genetically modified worms so that they express specific forms of a-syn in select nerve cells. These worms were engineered in such a way that defined neurons will start to fluoresce if transfer of a-syn has taken place. In the future, this worm model system and RNA interference can be used to screen in an unbiased way for molecular pathways that play important roles in a-syn transfer.PRISTINE-PD was aimed at understanding the molecular details of intercellular a-syn transfer and what role it plays in advancing the neuropathology in PD. Ultimately, the models developed in the project should define whether the prion-like process can be targeted to novel therapies that slow the progression of PD.