Dementia with Lewy bodies (DLB), Parkinson’s disease (PD) and multiple system atrophy are characterized by the deposition of disease-associated α-synuclein. In the present study we 1) examined the molecular specificity of the novel anti-α-synuclein 5G4 antibody; 2) evaluated immunoreactivity patterns and their correlation in human brain tissue with micro- and astrogliosis in 57 cases with PD or DLB; and 3) performed a systematic immunoelectron microscopical mapping of subcellular localizations. 5G4 strongly binds to the high molecular weight fraction of β-sheet rich oligomers, while no binding to primarily disordered oligomers or monomers was observed. We show novel localizations of disease-associated α-synuclein including perivascular macrophages, ependyma and cranial nerves. α-Synuclein immunoreactive neuropil dots and thin threads associate more with glial reaction than Lewy bodies alone. Astrocytic α-synuclein is an important component of the pathology. Furthermore, we document ultrastructurally the pathway of processing of disease-associated α-synuclein within neurons and astroglial cells. Interaction of mitochondria and disease-associated α-synuclein plays a key role in the molecular–structural cytopathogenesis of disorders with Lewy bodies. We conclude that 1) the 5G4 antibody has strong selectivity for β-sheet rich α-synuclein oligomers; 2) Lewy bodies themselves are not the most relevant morphological substrate that evokes tissue lesioning; 3) both neurons and astrocytes internalize disease-associated α-synuclein in the human brain, suggesting prion-like cell-to-cell spread of α-synuclein by uptake from surrounding structures, as shown previously in experimental observations.