Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons

Abstract

Early neuronal development is a well-coordinated process in which neuronal stem cells differentiate into polarized neurons. This process has been well studied in classical non-human model systems, but to what extent this is recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured human iPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. We found extensive remodeling of the neuron transcriptome and proteome, with altered mRNA expression of ~1,100 genes and different expression profiles of ~1,500 proteins during neuronal differentiation and polarization. We also identified a distinct stage in axon development marked by an increase in microtubule remodeling and apparent relocation of the axon initial segment from the distal to proximal axon. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons.(FOM, #16NEPH05, CJW). AUTHOR CONTRIBUTIONSFWL, RK and SP designed the research, conducted experiments, and wrote the article together with LJH. RS conducted and analyzed the mass spectrometry experiments supervised by MA, LJH performed and interpreted the electrophysiology data supervised by CJW, RK and BLS provided and interpreted the RNA sequencing analysis. HDM edited the article. CCH designed the experimental plan, supervised the research and wrote the article.

Centrosome‐mediated microtubule remodeling during axon formation in human iPSC‐derived neurons

“…To control for possible post‐differentiation effects of PLK4 inhibition unrelated to centriole number, we investigated if axonal targeting of Trim46 was affected when applying Centrinone‐B treatment after neuronal differentiation. Treating neurons with Centrinone‐B three days after neuronal induction, which we previously reported as a time point in which most neurons are differentiated but not yet polarized, did not affect Trim46 appearance at axons or at AnkG‐positive structures at later stages (Fig EV2L and M) (Lindhout et al, 2020). Together, these data suggest that centrosomes are important for the targeting of Trim46, but not AnkG, to axons during early stages of neuronal development.…”

“…Next, we aimed to quantify effects of centriole depletion during axon specification with unbiased profiling. Therefore, we performed mass spectrometry‐based quantitative proteomics analysis on days 1, 3, and 7, which generally corresponds with developmental stage 1, onset of stage 2, and onset of stage 3, respectively (Lindhout et al, 2020). We compared the proteome dynamics during early neurodevelopment of replicates of Centrinone‐B‐treated and control neurons (Fig EV3A, DATASET EV1).…”

“…To gain more insight into the potential role of centrosomes on the unique organization of the axonal microtubule cytoskeleton, marked by a uniform organization of plus‐end out microtubules, we studied the effect of centriole loss on the microtubule remodeling processes during development. We systematically analyzed plus‐end dynamics and orientations of microtubules in axons and dendrites on day 7 and 13, which coincide with the onset of stage 3 and late stage 3, respectively (Lindhout et al, 2020). For the analysis at axons, we measured both proximal and distal regions, as we previously reported that axon development follows a distal‐to‐proximal reorganization during this time window in human iPSC‐derived neurons (Lindhout et al, 2020).…”

“…To illustrate this, neurogenesis occurs after ~ 1 week in rodents, whereas this takes about ~ 3 months in humans, both in vivo and in vitro (Shi et al, 2012; Espuny‐Camacho et al, 2013; Otani et al, 2016; Sousa et al, 2017). This profound slower development of human iPSC‐derived neurons increases the temporal resolution to study axonal processes, which recently has led to the identification of an additional axon developmental stage in human neurons (Espuny‐Camacho et al, 2013; Lindhout et al, 2020; Otani et al, 2016; Linaro, 2019 #9). Altogether, this emphasizes the relevance of studying centrosome functions during axon development in human iPSC‐derived neurons as a model system.…”

Neuronal Microtubule Organization

“…The results obtained with these model organisms might not fully recapitulate the human situation. Neurons derived from human induced pluripotent stem cells (hiPSCs) could be a good way to overcome this problem and make a more relevant model to study human disorders and develop potential drugs (Lancaster et al, 2013;Lindhout et al, 2020;Meijer et al, 2019;Zhao and Bhattacharyya, 2018). In addition to iPSC-induced neurons, a glia-induced-neuron system exists, in which the repression of a single RNA binding polypyrimidine-tract-binding (PTB) protein can transdifferentiate mouse (in vivo) and human (in vitro) astrocytes into functional neurons (Qian et al, 2020;Xue et al, 2013).…”