Proteome dynamics in iPSC-derived human dopaminergic neurons

Dopaminergic neurons participate in fundamental physiological processes and are the cell type primarily affected in Parkinson"s disease (PD). Their analysis is challenging due to the intricate nature of their function, their involvement in diverse neurological processes, their heterogeneity and localization in deep brain regions. Consequently, most of the research on the protein dynamics of dopaminergic neurons has been performed in animal cells ex vivo. Here we use iPSC-derived, human mid-brain specific dopaminergic neurons to study general features of their proteome biology. In order to maximize the proteome coverage, we first performed in-depth identification of proteins in hDaN cultures by high-pH fractionation followed by measurement using Data-Independent Acquisition (DIA). With this approach, we cover the proteome to a depth of 9,409 proteins, which we estimate to present up to 80% of the total expressed cellular proteome. We next focused on investigation of protein turnover using dynamic SILAC labeling to measure the half-life of more than 4,300 proteins. We report uniform turnover rates of conserved protein complexes and identify several outliers in the mitochondrial outer membrane and mitophagy pathway.

We use differential dynamic SILAC labeling in combination with microfluidic devices to analyze local protein synthesis and transport between axons and soma. Briefly, neurons were seeded in a two-well microfluidic device (Xona Microfluidics) and pulsed with heavy amino acids in both wells simultaneously: Lys4+Arg6 in the somatodendritic well and Lys8+Arg10 the axonal well. Samples were harvested after different time points (12-120 hours), allowing temporal analyses of label incorporation. We report 105 potentially novel axonal markers and detect translocation of 269 proteins between axons and the soma in the time frame of our analysis (120 hours). Importantly, we provide evidence for local synthesis of 154 proteins in the axon and their retrograde transport to the soma, among them several proteins involved in RNA biology such as adenosine deaminase ADAR and RNA helicase DHX30, involved in the assembly of mitochondrial ribosomes. We are currently applying this workflow to investigate how oxidative stress, one of hallmarks of PD, influences protein synthesis, trafficking and turnover. Our study provides a workflow and resource for future applications of quantitative proteomics in iPSC-derived human neurons.