Under normal conditions, α-synuclein (αS) is preferentially localized within synaptic boutons. Changes in αS localization may occur, however, as a consequence of a variety of pathophysiological conditions that include increased protein expression. The purpose of this study was to investigate intraneuronal sites of overexpression-induced αS accumulation. In particular, we assessed αS burden within neuronal perikarya and nuclei; the latter analysis was facilitated by a newly developed assay that detected interactions between αS and histones.Experiments were carried out in the substantia pars compacta of rats treated with a unilateral injection of AAVs delivering human αS DNA. Midbrain tissue sections were stained to detect neuronal αS immunoreactivity. Nuclear αS localization was evaluated in situ using a proximity ligation assay (PLA). Immunoprecipitation was carried out to detect αS-histone interactions in nuclear preparations from rat midbrain tissue. αS was undetectable in the cytosol and nuclei of nigral neurons from non-injected control animals. Quite in contrast, midbrain tissue sections processed for immunohistochemistry showed that overexpression of human αS was associated with robust accumulation of the exogenous protein within both neuronal perikarya and nuclei. PLA not only detected overexpression-induced nuclear αS with great sensitivity and specificity but also indicated interactions of human αS with histones. αS-histone interactions were confirmed biochemically by immunoprecipitation using midbrain nuclei. Our results provide evidence of a relationship between increased αS expression, cytosolic protein accumulation and αS access into neuronal nuclei. Data also reveal that, once in the nucleus, αS closely interacts with histones, indicating a mechanism of likely pathophysiological relevance.