Updated project metadata. Alpha-Synuclein (α-Syn) association with exosomes has been extensively studied both as a physiological process but also as a means of disease transmission under pathological conditions, via an elusive mechanism. Here, we utilized the preformed fibril (PFF) mouse model, as a source of brain-derived exosomes and assessed their pathogenic capacity following intrastriatal injections in host wild type (WT) mouse brain. We further investigated the impact of PFF toxic stimulus in the exosomal cargo independent of the endogenous α-Syn, by isolating exosomes from PFF-injected α-Syn knockout mice. We found that PFF inoculation does not alter the morphology, size distribution, and quantity of brain-derived exosomes, however we show, for the first time, that it triggers changes in the exosomal protein cargo related to synaptic and mitochondrial function, as well as metabolic processes. Importantly, we showed that the presence of the endogenous α-Syn is essential for the exosomes to acquire a pathogenic identity/status, allowing them to mediate disease transmission by inducing phospho-α-Syn pathology, astrogliosis and synaptic alterations in the host mouse brain, thus supporting a role of exosomes in a prion-like mode of infection.