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Abstract
Developing therapeutic approaches that target neuronal differentiation will be greatly beneficial for the regeneration of neurons and synaptic networks in neurological diseases. Protein synthesis (mRNA translation) has recently been shown to regulate neurogenesis of neural stem/progenitor cells (NSPCs). However, it has remained unknown whether engineering translational machinery is a valid approach for manipulating neuronal differentiation. The present study identifies that a bivalent securinine compound SN3-L6, previously designed and synthesized by our group, induces potent neuronal differentiation through a novel translation-dependent mechanism. An isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis in Neuro-2a progenitor cells revealed that SN3-L6 upregulated a group of neurogenic transcription regulators, and also upregulated proteins involved in RNA processing, translation, and protein metabolism. Notably, puromycylation and metabolic labeling of newly synthesized proteins demonstrated that SN3-L6 induced rapid and robust activation of general mRNA translation. Importantly, mRNAs of the proneural transcription factors Foxp1, Foxp4, Hsf1, and Erf were among the targets that were translationally upregulated by SN3-L6. Either inhibition of translation or knockdown of these transcription factors blocked SN3-L6 activity. We finally confirmed that protein synthesis of a same set of transcription factors was upregulated in primary cortical NPCs. These findings together identify a new compound for translational activation and neuronal differentiation, and provide compelling evidence that reprogramming transcriptional regulation network at translational levels is a promising strategy for engineering NSPCs.
Highlights
Neuronal differentiation, including neurogenesis and the following neurite outgrowth, is a highly dynamic process that requires biogenesis of a series of critical proteins to achieve the transitions through different cellular states
The following primary antibodies and inhibitors were used: antibodies against β-tubulin III and α-tubulin were purchased from Sigma-Aldrich; Antibodies against puromycin and MAP2 were from Millipore; Rabbit monoclonal antibodies against Foxp1, heat shock transcription factor 1 (Hsf1), β-actin, total or phosphorylated forms of mTOR, S6K, S6, 4EBP1, eEF2K, eEF2, and eIF2α were from Cell Signaling Technology (Beverly, MA, United States); Foxp4, ETS2 repressor factor (Erf), and GAPDH antibodies were from Abcam (Cambridge, United Kingdom)
By examining different fractions of ribonucleoprotein, monosome and polysome, we found that mRNAs of Foxp1, Foxp4, Hsf1, and Erf all showed a shift of distribution to heavier polysome fractions (Figures 6M–P), which indicates that these mRNAs are more translational active in SN3-L6-treated cells
Summary
Neuronal differentiation, including neurogenesis and the following neurite outgrowth, is a highly dynamic process that requires biogenesis of a series of critical proteins to achieve the transitions through different cellular states. Increasing evidence has suggested that translational regulation, including mRNA localization, ribosome biogenesis and the translation initiation or elongation control, is another critical way to remodel the transcription factor network for neuronal differentiation (Kusek et al, 2012; Vessey et al, 2012; Hartman et al, 2013; Kraushar et al, 2013, 2014; Roffe et al, 2013; MacNicol et al, 2015; Werner et al, 2015; Popovitchenko et al, 2016) These recent findings lead to a possible alternative strategy for inducing or manipulating neuronal differentiation, which is to target translation-dependent regulation of transcription factors or other cell fate determinants. Despite the potential advantage that translation probably leads to quicker and more efficient control of protein levels without modifications of the host genome, it has not been reported far whether chemical induction of translation by small molecules is possible to promote neuronal differentiation
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