Updated project metadata. Brain aggregates of α-synuclein (α-syn) characterizes a group of diseases defined as synucleinopathies. Recent studies implicate neuroinflammation as one of the central pathogenic mechanisms. However, how inflammation is linked to the α-syn pathology is still unknown. We wanted to address this by a ‘double-hit’ approach. Three weeks after intra-striatal injections of human α-syn pre-formed fibrils (PFF), mice were subjected for 3 weeks to repeated intraperitoneal injections of low concentrations (1 mg/ml) of lipopolysaccharide (LPS), a component of gram-negative bacteria. Histological analysis confirmed brain propagation of α-syn aggregation in PFF-injected mice independent of LPS-injections. No motor dysfunction was observed at this stage as measured in the pole test. Spleen immune cell profiling and multiplex cytokine analysis confirmed LPS-induced changes in T- and B-cells populations, monocytes, and neutrophils, and increased brain TNF-α, IL-β, IL-10 and KC/GRO levels. LC-MS/MS analysis in the forebrain area and subsequent downstream ReactomeGSA pathway analysis revealed that PFF-injections induced alterations in mitochondrial metabolism and neurotransmitter signaling. Systemic inflammation resulted in an overrepresentation of complement and coagulation pathways and upregulation of integrin and B cell receptor signaling in the PFF-injected mice.