Human cortical organoids expose a differential function of GSK3 on direct and indirect neurogenesis

Abstract

The regulation of proliferation and polarity of neural progenitors is crucial for the development of the brain cortex, with modes and timings of cell division intimately related to the stereotypical acquisition of layerspecific neuronal identities. Animal studies have implicated glycogen synthase kinase 3 (GSK3) as a pivotal regulator of both proliferation and polarity, yet the functional relevance of its signaling for the unique features of human corticogenesis remain to be elucidated. Here we harness human cortical brain organoids to probe the longitudinal impact of GSK3 inhibition through multiple developmental stages. Our results indicate that chronic GSK3 inhibition increases the proliferation of neural progenitors and causes massive derangement of cortical tissue architecture. Surprisingly, single cell transcriptome profiling revealed only a discrete impact on early neurogenesis and uncovered a pivotal role of GSK3 in the regulation of NEUROD1/2 lineages and outer radial glia (oRG) output, without compromising direct neurogenic trajectories. Through this first single cell-level dissection of the GSK3 regulatory network in human corticogenesis, our work underscores the robustness of transcriptional programs in determining neuronal identity independent of tissue architecture. IntroductionNeurogenesis is initiated by the formation of a neuroepithelium, composed by neural stem cells (NSCs) organized in apico-basal orientation that divide symmetrically leading to a rapid expansion of the NSC pool (Taverna et al., 2014;Temple, 2003). The polarization of the neuroepithelium precedes the differentiation of NSCs into radial glia cells (RGCs), and triggers the elongation of cytoplasmic processes that form a migratory scaffold for newborn neurons (Betizeau et al., 2013). Such polarization is in turn necessary for the acquisition of the key properties that define tissue organization (Johansson et al., 2010).Glycogen synthase kinase 3 alfa and beta (GSK3 and ) are serine/threonine kinases encoded by 2 different genes, which function as integrating hubs for multiple proliferation and differentiation signals due to their central role in the receptor tyrosine kinase, Wnt and sonic hedgehog signaling pathways McCubrey et al., 2016). GSK3 is involved in neurodevelopment through the phosphorylation of a broad set of substrates, including transcription factors essential for brain development, such as CREB (Grimes and Jope, 2001a), neurogenin2 (Ma et al., 2008) β-catenin (Aberle et al., 1997 and multiple microtubule-associated proteins (Fumoto et al., 2008;Zhou et al., 2004). Studies in animal models have provided a wealth of evidence linking GSK3 activity to the regulation of early and late neurogenesis in a stage-wise fashion. Its activity is required to maintain the overall polarity of the radial glia scaffold (Yokota et al., 2010). Genetic ablation of both GSK3 or GSK3 in radial glial cells (RGCs) results in a massive increase in neural progenitor proliferation with marked suppression of intermediate progenitor cells (...

Investigating How Inflammation Involving NF-κB Signaling Disrupts the Genetic Architecture of Neurons/Neural Stem Cells and Fuels Neurodegeneration

“…Proper Wnt signaling is crucial for maintaining neurogenesis and preventing excessive neuronal death. GSK3 beta damage may disrupt synaptic plasticity, a key process for learning and memory [342,343]. Altered In ammatory conditions may compromise the survival signaling cascades regulated by LHX transcription factors.…”