Abstract:
Dopaminergic neurons are a small but essential neuronal population located primarily in the substantia nigra, ventral tegmental area, and hypothalamus. They play central roles in motor control, motivation, reward, and higher-order cognition. Their selective vulnerability, particularly in Parkinson’s disease, highlights the importance of understanding their biology at the molecular level. However, human dopaminergic neurons are heterogeneous and difficult to access, making their analysis challenging.
I present a comprehensive analysis of the proteome and protein dynamics of human midbrain dopaminergic neurons derived from iPSC. Using label-free and dynamic SILAC-based proteomics, I quantified over 9,400 proteins and determined the half-lives of more than 4,300 proteins. The results revealed patterns of proteome organization: while cytosolic protein complexes such as the proteasome displayed uniform turnover kinetics, mitochondrial respiratory chain complexes exhibited marked heterogeneity in their stability and replacement rates. I combined dynamic SILAC with microfluidic devices that separated neuronal axons from their soma to study compartment-specific proteomes. This approach allowed direct measurement of local protein synthesis, degradation, and trafficking in axons. I identified 105 novel axonal markers, detected the dynamic redistribution of 269 proteins between soma and axons over a 120-hour time course, and, importantly, demonstrated local axonal translation of 154 proteins. Several of these proteins, including ADAR1 and DHX30, were implicated in RNA editing and mitochondrial ribosome biogenesis, highlighting an unanticipated coupling between axonal translation and mitochondrial function. These datasets provide a resource for probing the mechanisms of neuronal compartmentalization and vulnerability.
I also examined how mitochondrial stress impacted proteome homeostasis in human dopaminergic neurons. Chronic low-dose exposure to rotenone, a mitochondrial complex I inhibitor, caused significant remodeling of the neuronal proteome. Dynamic SILAC showed an overall increase in protein half-lives, indicating a slowdown of new protein synthesis under stress conditions. Notably, mitochondrial respiratory complexes and ROS detoxification pathways exhibited distinct changes in abundance, with selective upregulation of antioxidant enzymes such as SOD2. However, the accumulation of mitochondrial protein dysregulation did not trigger mitochondrial clearance pathways. These findings offer mechanistic insight into how dopaminergic neurons maladaptively respond to mitochondrial dysfunction, a hallmark of Parkinson’s disease pathogenesis.
In summary, this thesis integrates global, dynamic, and spatial proteomics to characterize the proteome biology of iPSC-derived human dopaminergic neurons, and provides datasets that can serve as a foundation for future studies.
dopaminergic neurons