Parkinson’s disease (PD) is a synucleinopathy that has multiple neuropathological characteristics, with nigrostriatal dopamine system degeneration being a core feature. Current models of PD pathology typically fail to recapitulate several attributes of the pathogenic process and neuropathology. We aimed to define the effects of combining a mouse model exhibiting multiple PD-like changes with intrastriatal injections of α-synuclein (α-syn) pre-formed fibril (PFFs) aggregates. We employed the heterozygous Engrailed 1 (En1+/–) mouse that features several pathophysiological hallmarks of clinical PD. To test the hypothesis that the neuropathological changes in the En1+/–mice will promote formation of α-syn aggregates following intrastriatal injections of pathogenic human α-syn PFFs. We unilaterally injected PFFs into the striata of 1-month-old En1+/–and control wild-type mice and euthanized animals at 3 months for post-mortem analysis. Using immunohistochemistry and unbiased stereology, we established that PFF-injected En1+/–mice exhibited a near-threefold increase in pS129-α-syn-positive neurons in the substantia nigra compared to PFF-injected wild-type mice. The PFF-injected En1+/–mice also displayed significant increases in pS129-α-syn-positive neurons in the amygdala and ventral tegmental area; regions of known PD pathology with projections to the striatum. Additionally, we observed amplified pS129-α-syn-positive aggregation in En1+/–mice in multiple cortical regions. Following intrastriatal injection of PFFs, absence of an En1allele leads to additional aggregation of pathological α-syn, potentially due to En1-loss mediated nigrostriatal impairment. We propose that further development of this double-hit model could result in a PD mouse model that predicts which experimental therapies will be effective in PD.