Feline Lindhout

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons 2 33 1988). These neurons undergo five well-defined developmental stages, transforming from round, spherical cells to fully mature, polarized neurons (Craig and Banker 1994). First, the symmetric young neuron forms small processes (stage 1) and multiple neurites (stage 2). Next the cells undergo polarization, where one neurite is specified as the axon (stage 3), while the remaining neurites will further develop into dendrites. The axon rapidly extends and further matures by the formation of the axon initial segment (AIS) (Leterrier 2018). The AIS is required for generating action potentials (APs) and maintaining neuronal polarity. In addition to the classic AIS component Ankyrin-G (AnkG), the microtubule binding protein Trim46 also localizes to the AIS and is critical for axon formation by forming parallel microtubule bundles in the proximal axon (van Beuningen et al. 2015; Gumy et al. 2017; Harterink et al. 2018). As the neuron matures, the developing axons and dendrites undergo significant morphological and molecular changes and form dendritic spines (stage 4-5), which allow for the formation of synaptic contacts and the establishment of functional neuron-to-neuron interactions (Harris and Kater 1994; Fletcher, De Camilli, and Banker 1994; Grabrucker et al. 2009). In depth proteomic analysis of primary rat neurons in culture have identified a number of specific pathways and unique protein profiles that contribute to various aspects of neurodevelopment processes (Frese et al. 2017). Proper characterization and quantitative profiling of transcriptome and proteome dynamics is essential to study the specific neurodevelopment events in human iPSC-derived neuronal cultures, including early developmental changes such as neuronal polarization and axon specification. In this study, we performed extensive characterization of the early developmental stages of hiPSC-derived neurons by immunocytochemistry, electrophysiology, RNA sequencing, and stable isotope labeling combined with high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). We established transcriptomic and proteomic profiles of the early developmental stages (stage 1-3), comprising 14,551 transcripts and 7,512 protein identifications, of which we assessed 1,163 and 2,218 factors that showed differential expression, respectively. These transcriptomic and proteomic profiles point to the importance of microtubule cytoskeleton remodeling in the early stage of neuronal development. Combining this framework with additional methods such as genetic manipulation and live-cell imaging allowed us to investigate the cellular and molecular processes during neuronal polarization and axon outgrowth. Specifically, we identified a distinct, previously unrecognized developmental stage during early axon development, characterized by the reorganization of the axonal microtubule network and relocation of AIS proteins from the distal to proximal axon. The transition through these early axon developmental stages coincided with the time window in which maturation of action potentials occurred. Together, our study provides a quantitative description of transcriptomic and proteomic profiles of hiPSC-derived neuron cultures, which is a rich resource for further analyses of critical signaling pathways during early human neurodevelopment.