SummaryThe microenvironment of developing neurons is a dynamic landscape of both chemical and mechanical cues that regulate cell proliferation, differentiation, migration, and axon extension. While the regulatory roles of chemical ligands in neuronal morphogenesis have been described, little is known about how mechanical forces influence neurite development. Here, we tested how substratum elasticity regulates neurite development of human forebrain (hFB) neurons and human motor neurons (hMNs), two populations of neurons that naturally extend axons into distinct elastic environments. Using polyacrylamide and collagen hydrogels of varying compliance, we find that hMNs preferred rigid conditions that approximate the elasticity of muscle, whereas hFB neurons preferred softer conditions that approximate brain tissue elasticity. More stable leading-edge protrusions, increased peripheral adhesions, and elevated RHOA signaling of hMN growth cones contributed to faster neurite outgrowth on rigid substrata. Our data suggest that RHOA balances contractile and adhesive forces in response to substratum elasticity.
Patients with Tuberous Sclerosis Complex (TSC) show aberrant wiring of neuronal connections formed during development which may contribute to symptoms of TSC, such as intellectual disabilities, autism, and epilepsy. Yet models examining the molecular basis for axonal guidance defects in developing human neurons have not been developed. Here, we generate human induced pluripotent stem cell (hiPSC) lines from a patient with TSC and genetically engineer counterparts and isogenic controls. By differentiating hiPSCs, we show that control neurons respond to canonical guidance cues as predicted. Conversely, neurons with heterozygous loss of TSC2 exhibit reduced responses to several repulsive cues and defective axon guidance. While TSC2 is a known key negative regulator of MTOR-dependent protein synthesis, we find that TSC2 signaled through MTOR-independent RHOA in growth cones. Our results suggest that neural network connectivity defects in patients with TSC may result from defects in RHOA-mediated regulation of cytoskeletal dynamics during neuronal development.
Growth cones at the tips of extending axons navigate through developing organisms by probing extracellular cues, which guide them through intermediate steps and onto final synaptic target sites. Widespread focus on a few guidance cue families has historically overshadowed potentially crucial roles of less well-studied growth factors in axon guidance. In fact, recent evidence suggests that a variety of growth factors have the ability to guide axons, affecting the targeting and morphogenesis of growth cones in vitro. This review summarizes in vitro experiments identifying responses and signaling mechanisms underlying axon morphogenesis caused by underappreciated growth factors.
Growth cones at the distal tips of developing axons transduce extracellular chemoattractants and repellents into guided motility throughout developing organisms. Repulsive factors can produce abrupt cytoskeletal contraction in growth cones, coupled with internalization of plasma membrane in a process called “collapse”. Using neurons differentiated from induced pluripotent stem cells (iPSCs), we examine mechanisms that control normal human cortical neuron development and dysregulation of these signals in Tuberous Sclerosis Complex (TSC). TSC is a neurodevelopmental disorder caused by pathological variants of the genes encoding TSC1 or TSC2, resulting in the formation of cortical tubers and improper neuronal connectivity. These defects likely contribute to TSC neuropathology such as cognitive deficits, autism, and epilepsy. Using immunoassays and live cell imaging, our lab has shown that TSC2+/− neurons fail to respond the chemorepellent EphrinA1 in several quantifiable measures. TSC2+/− growth exhibit reduced rapid membrane internalization via macropinocytosis and show less elevated receptor tyrosine kinase signaling, as well as reduced cytoskeletal contraction and F‐actin depolymerization. Studies have shown that endocytosis and trafficking of receptor tyrosine kinase (RTK) signaling endosomes are required to elicit a proper guidance response. Immunoassays and live endosome imaging in TSC2+/− growth cones suggest normal EphrinA1‐EphA4 internalization, but defective endosome trafficking of EphrinA1 from early endosomes to late endosomes. Other experiments implicate defects in growth cone microtubule dynamics, a poorly understood but essential process for proper endosome trafficking and guidance cue responses. This research aims to improve our understanding of RTK endosome trafficking and show how defects in these processes can lead to neurodevelopmental disorders.
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