Single nucleus RNA sequencing analysis combined with in vitro validation in human dopaminergic neurons reveals aspects of idiopathic Parkinson's disease pathology

The number of cases of Idiopathic Parkinson's disease (IPD) has doubled in the past 25 years, with this increase expected to continue its rapid incline, as the population ages. During the disease"s onset, a variety of clinical symptoms gradually arise, including both motor and nonmotor, the latter manifesting earlier, before an observation of the motor symptoms or an exact diagnosis can be made. Nonetheless, our understanding of the exact molecular mechanisms associated with the generation and progression of this disease has yet to be fully understood. To gain detailed insights into IPD cellular pathology, we utilized a publicly available dataset and analyzed 41,435 single-nuclei transcriptomes of post-mortem midbrain from IPD patients (n=6) and healthy donors (n=5). Overall, alteration in a plethora of subtypes was observed, with oligodendrocytes, microglia, and dopaminergic neurons (DANs) being the more substantial ones. Differential analysis of activated transcription factors revealed dysregulation of dopaminergic-related TFs in IPD samples, with NR4A2 being one of the most evident ones. Similarly, Trail and p53 pathways were significantly altered, indicating a malfunction in pathways associated with cell death. To validate these results in vitro, we generated an IPD-like cellular model of human DANs that inducibly expressed the EYFP-SNCA WT transgene. Specifically, human neural progenitor cells (hNPCs) were stably transfected with the TetOn EYFP-SNCA WT cassette and directly differentiated into midbrain dopaminergic neurons. Interestingly, the generated DANs exhibited high levels of tyrosine hydroxylase (TH), dopamine transporter (SLC6A3), and vesicular monoamine transporter 2 (SLC18A2). Additionally, they were characterized by the absence of lipoprotein lipase (LPL) and the increased expression of Aldehyde Dehydrogenase 1 (ALDH1A1), a pattern specific to IDP-prone DaNs. After inducible expression of EYFP-SNCA WT for 5 days, DaNs exhibited a significant activation of apoptosis as well as impairment in the patterns of transcription factors involved in dopaminergic neuron maintenance, particularly that of NR4A2, which was also found to be dysregulated in our computational analysis. Interestingly, in silico knockout of targeted key players had a significant impact on IPD-related neurodegeneration, indicating potential therapeutic interventions. Our findings propose a potent workflow where a combination of snRNA-seq analysis along with the in vitro validation in a reliable cellular model might reveal crucial aspects of IPD pathology.